專利名稱：在棒狀細(xì)菌中可自主復(fù)制的質(zhì)粒的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及一種來源于熱產(chǎn)氨棒狀桿菌的新質(zhì)粒。本發(fā)明的質(zhì)?？梢杂糜诟倪M(jìn)棒狀細(xì)菌，所述的棒狀細(xì)菌用作生產(chǎn)如L-氨基酸這樣的有用物質(zhì)的細(xì)菌。
包括L-谷氨酸和L-賴氨酸在內(nèi)的氨基酸是采用所謂的棒狀細(xì)菌或其變體菌株通過發(fā)酵的方法生產(chǎn)的，其中所述的棒狀細(xì)菌通常屬于短桿菌屬、棒狀桿菌屬或微桿菌屬(《氨基酸發(fā)酵》Gakkai Shuppan中心，第195-215頁，1986)。
在氨基酸的工業(yè)發(fā)酵生產(chǎn)中，除了在相對于糖的產(chǎn)量有所提高、縮短培養(yǎng)時(shí)間、氨基酸積累的濃度提高等等之外，采用升高的培養(yǎng)溫度作為提高經(jīng)濟(jì)效率的一個(gè)技術(shù)因素被認(rèn)為是重要的。即通常在最佳的發(fā)酵溫度下進(jìn)行培養(yǎng)，并且對谷氨酸棒狀桿菌而言最佳的溫度為31.5℃。在開始培養(yǎng)后，發(fā)酵過程中產(chǎn)生了熱量如果不移除該熱量，會(huì)使氨基酸的生產(chǎn)顯著下降。所以，為了維持培養(yǎng)肉湯的溫度為最佳值，需要冷卻設(shè)備。另一方面，如果可以升高培養(yǎng)溫度的話，那么減少冷卻所需的能量是可能的，并且可以使冷卻設(shè)備的變小。
在棒狀細(xì)菌中，已經(jīng)分離出熱產(chǎn)氨棒狀桿菌作為可以在高溫區(qū)中生長的棒狀細(xì)菌(日本專利申請延遲(公開)號63-240779)、雖然谷氨酸棒狀桿菌的生長在40℃下受到了明顯的抑制，但熱產(chǎn)氨棒狀桿菌可以在大約40℃或者更高的溫度下生長并且被認(rèn)為適合于高溫發(fā)酵。
目前，依賴于DNA重組技術(shù)的育種正在大腸桿菌或棒狀細(xì)菌中開展著。為了通過DNA重組技術(shù)改進(jìn)微生物，甚至常常使用來源于屬于另一個(gè)種或?qū)俚奈⑸锘蛘邔挼乃拗鬏d體譜的質(zhì)粒。但是，通常使用適合于育種目的微生物的質(zhì)粒。具體地講，當(dāng)對育種的目的微生物而言的最佳培養(yǎng)溫度不同于同一個(gè)種或?qū)俚奈⑸锏淖罴褱囟葧r(shí)，采用適合于所述微生物的質(zhì)粒是優(yōu)選的。
迄今獲得的來源于棒狀細(xì)菌的質(zhì)粒為來自乳發(fā)酵短桿菌ATCC13869的pAM330(日本專利申請延遲(公開)號58-67669)、來自乳發(fā)酵短桿菌ATCC21798的pBL1(Santamaria.R.等人《遺傳微生物學(xué)雜志》130，第2237-2246頁，1984)、來自谷氨酸棒狀桿菌ATCC13058的pHM1519(日本專利申請延遲(公開)號58-77895)、來自谷氨酸棒狀桿菌ATCC31808的pCG1(日本專利申請延遲(公開)號57-134500)以及來自谷氨酸棒狀桿菌DSM58的pGA1(日本專利申請延遲(公開)號9-2603011)。
但是，目前尚未獲得適合于熱產(chǎn)氨棒狀桿菌的質(zhì)粒。
本發(fā)明的一個(gè)目的在于提供一種對改進(jìn)可以在升高的溫度下生長的棒狀細(xì)菌-熱產(chǎn)氨棒狀桿菌(Corynebacterium thermoaminogenes)有用的質(zhì)粒。
本發(fā)明的發(fā)明人發(fā)現(xiàn)熱產(chǎn)氨棒狀桿菌AJ12340(FERM BP-1539)、AJ12308(FERM BP-1540)、AJ12309(FERM BP-1541)、AJ12310(FERMBP-1542)各自攜帶適合于各菌株的隱蔽性質(zhì)粒，并且成功地分離和鑒定了各個(gè)質(zhì)粒。于是，他們完成了本發(fā)明。
即本發(fā)明提供了一種可以從熱產(chǎn)氨棒狀桿菌中分離的質(zhì)?；蚱溲苌?，該質(zhì)粒包含編碼Rep蛋白的基因(rep基因)并且大小約為4.4kb或約為6kb，所述的Rep蛋白具有SEQ ID NO2中所示的氨基酸序列或具有與前述氨基酸序列90％或更高同源性的氨基酸序列。
前面提到的質(zhì)粒的例子包括可以從熱產(chǎn)氨棒狀桿菌AJ12340(FERMBP-1539)、AJ12308(FERM BP-1540)或AJ12310(FERM BP-1542)中分離的質(zhì)粒，該質(zhì)粒的大小約為4.4b并且由


圖1中所示的限制圖表示，以及可以從熱產(chǎn)氨棒狀桿菌AJ12309(FERM BP-1541)中分離的質(zhì)粒，該質(zhì)粒的大小約為6kb并且由圖2中所示的限制圖表示。
前面提到的質(zhì)粒的具體例子包括包含編碼具有SEQ ID NO2、4或6中所示氨基酸序列的Rep蛋白之基因的質(zhì)粒，以及包含編碼具有SEQ IDNO8中所示氨基酸序列的Rep蛋白之基因的質(zhì)粒。
圖1為本發(fā)明的質(zhì)粒pYM1，pYM2和pYM3的限制圖。
圖2為本發(fā)明的質(zhì)粒pYM4的限制圖。
圖3顯示出pYMFK的構(gòu)建。
圖4顯示出pYMK的構(gòu)建。
圖5顯示出pYMC的構(gòu)建。
圖6顯示出pK1的構(gòu)建。
按照用于制備質(zhì)粒的常用方法、比如堿法(《生物工程實(shí)驗(yàn)教材》由日本生物科學(xué)與生物工程協(xié)會(huì)編輯，第105頁，Baifukan，1992)，可以從熱產(chǎn)氨棒狀桿菌AJ12340(FERM BP-1539)、AJ12308(FERM BP-1540)、AJ12309(FERM BP-1541)或AJ12310(FERM BP-1542)中分離出本發(fā)明的質(zhì)粒。對FERM BP-1539來說，遵照布達(dá)佩斯條約的條款于1987年10月27日將其在1987年3月13日在工業(yè)科學(xué)與技術(shù)局國立生物科學(xué)與人體技術(shù)研究院(郵政編碼305-8566，1-3 Higashi 1-chome，Tsukuba-shi，Ibaraki-ken，日本)中的原始保藏(保藏號為FERM P-9277)轉(zhuǎn)變?yōu)閲H保藏，并已保藏在同一保藏機(jī)構(gòu)中。對FERMBP-1540、FERMBP-1541和FERM BP-1542來說，遵照布達(dá)佩斯條約的條款于1987年10月27日將它們在1987年3月10日在前面提到的保藏機(jī)構(gòu)中的原始保藏(保藏號為FERM P-9244、FERM P-9245和FERM P-9246)轉(zhuǎn)變?yōu)閲H保藏，并已保藏在同一保藏機(jī)構(gòu)中。
本發(fā)明的發(fā)明人從前面提到的熱產(chǎn)氨棒狀桿菌AJ12308(FERMBP-1540)、AJ12310(FERM BP-1542)、AJ12340(FERM BP-1539)和AJ12309(FERM BP-1541)中分離并鑒定了各自適合于各種所述細(xì)菌的質(zhì)粒，并依次命名為pYM1、pYM2、pYM3和pYM4。這些質(zhì)粒為在熱產(chǎn)氨棒狀桿菌的細(xì)胞中以雙鏈環(huán)狀DNA的形式存在的質(zhì)粒。包含于pYM1中的rep基因的核苷酸序列示于SEQ ID NO1中，包含于pYM2中的rep基因的核苷酸序列示于SEQ ID NO3中，包含于pYM3中的rep基因的核苷酸序列示于SEQ ID NO5中，且包含于pYM4中的rep基因的核苷酸序列示于SEQ ID NO7中?？梢杂砂谶@些質(zhì)粒中的rep基因編碼的氨基酸序列示于SEQ ID NOS2，4，6和8中。pYM1、pYM2和pYM3各自的大小約為4.4kb。pYM4的大小約為6kb。
在表1中顯示出當(dāng)用典型的限制酶消化pYM1、pYM2和pYM3時(shí)可以獲得的片段的數(shù)目和大小。在表2中顯示出當(dāng)用典型的限制酶消化pYM4時(shí)可以獲得的片段的數(shù)目和大小。此外，pYM1、pYM2和pYM3的限制圖示于
圖1中，pYM4的限制圖示于圖2中。
表1限制酶消化位點(diǎn)的數(shù)目 DNA片段(kb)BglⅡ 0 -BamHⅠ 2 1.8，2.6BstPⅠ 1 4.4EcoRⅠ 1 4.4HincⅡ 4 0.3，0.5，2.0，1.6HindⅢ 0 -KpnⅠ 0 -NaeⅠ 2 0.1，4.3NcoⅠ 1 4.4NheⅠ 2 1.8，2.6PmaCⅠ 1 4.4SacⅠ 0 -SalⅠ 0 -SacⅡ 3 0.1，1.4，2.9SmaⅠ 3 0.1，1.8，2.5SphⅠ 0 -TthlllⅠ 1 4.4XbaⅠ 0 -
表2限制酶消化位點(diǎn)的數(shù)目 DNA片段(kb)BglⅡ16.0BamHⅠ 23.8，2.2BstPⅠ 21.2，4.8EcoRⅠ 16.0HincⅡ 40.3，0.4，1.2，1.7，2.4HindⅢ 0-KpnⅠ0-NaeⅠ20.1，5.9NcoⅠ30.2，2.8，3.0NheⅠ30.1，2.3，3.6PmaCⅠ 0-SacⅠ0-SalⅠ0-SacⅡ50.1，0.2，0.9，1.8，3.0SmaⅠ20.1，5.9SphⅠ0-TthlllⅠ 0-XbaⅠ0-對本發(fā)明質(zhì)粒的核苷酸序列的測定表明pYM1、pYM2和pYM3分別含有4368bp、4369bp和4369bp，在核苷酸序列的水平上它們具有基本相同的結(jié)構(gòu)并且顯示出彼此之間具有99.9％的同源性。此外，pYM4含有5967bp，對于除大約1.6kb區(qū)域之外的約4.4kb的區(qū)域而言，它顯示出與pYM1、pYM2和pYM3之間具有極高的同源性，而其整體與它們之間顯示出大約81％的同源性。
所述質(zhì)粒含有彼此之間顯示出高度同源性的各自的rep基因。針對由rep基因編碼的Rep蛋白之氨基酸序列(SEQ ID NOS2，4，6和8)以及由來源于棒狀細(xì)菌的已知質(zhì)粒之rep基因編碼的Rep蛋白之氨基酸序列來比較同源性。在pYM1、pYM2和pYM3之間觀察到99％或者更高的同源性，在pYM2和pYM4之間觀察到81.91％的同源性。另一方面，它們顯示出與棒狀細(xì)菌的已知質(zhì)粒pAM330之間沒有同源性，并且它們顯示出與pGA1和pCG1之間具有80％或者更低的同源性。結(jié)果示于表3中。因此，在Rep蛋白的同源性的基礎(chǔ)上，本發(fā)明的質(zhì)粒與已知的棒狀細(xì)菌的質(zhì)粒是可以進(jìn)行區(qū)別的。
根據(jù)Takashi，k.和Gotoh，O.的方法，見J.Biochem.，92，1173-1177(1984)來計(jì)算同源性。
表3由不同的質(zhì)粒編碼的Rep蛋白之氨基酸序列的同源性pYM2 pYM4 pGA1 pCG1pYM2- 81.91％ 68.01％ 70.73％pYM4- - 69.39％ 70.23％pGA1- - - 75.31％pCG1- - - -由于本發(fā)明的質(zhì)粒可以在包括熱產(chǎn)氨棒狀桿菌在內(nèi)的棒狀細(xì)菌的細(xì)胞中充分地復(fù)制，因此通過在任意位點(diǎn)插入質(zhì)粒或其衍生物的外源基因并且用獲得的重組質(zhì)粒轉(zhuǎn)化宿主微生物，可以在宿主微生物中表達(dá)外源基因的遺傳信息。
下面列舉了棒狀細(xì)菌的例子嗜乙酰乙酸棒狀桿菌、醋谷棒狀桿菌、美棒狀桿菌、谷氨酸棒狀桿菌、熱產(chǎn)氨棒狀桿菌、百合花棒狀桿菌(谷氨酸棒狀桿菌)、Corynebacterium melassecola、擴(kuò)展短桿菌(谷氨酸棒狀桿菌)、乳發(fā)酵短桿菌(谷氨酸棒狀桿菌)、解糖短桿菌、Brevibacteriumimmariophilum、玫瑰色短桿菌、黃色短桿菌(谷氨酸棒狀桿菌)、生硫短桿菌。
本發(fā)明質(zhì)粒的衍生物意指由本發(fā)明質(zhì)粒的一部分組成的質(zhì)粒、或者由本發(fā)明質(zhì)粒的一部分或本發(fā)明的質(zhì)粒與另一種DNA序列組成的質(zhì)粒。部分意指含有對質(zhì)粒的自主復(fù)制來說必需的區(qū)域的部分。即使缺失了除對質(zhì)粒的自主復(fù)制來說必需的區(qū)域之外的區(qū)域(復(fù)制控制區(qū))、即除了含有復(fù)制起點(diǎn)和對復(fù)制而言必需的基因的區(qū)域之外的區(qū)域，本發(fā)明的質(zhì)粒仍然可以在宿主微生物中復(fù)制。此外，包括這種缺失的質(zhì)粒具有更小的尺寸。因此，具有這種缺失的質(zhì)粒優(yōu)選用作載體。再者，如果將諸如抗藥性基因這樣的標(biāo)記基因插入到本發(fā)明的質(zhì)?；蚱洳糠种械脑?，由于轉(zhuǎn)化體中標(biāo)記基因的表型，從而變得易于檢測轉(zhuǎn)化體?？梢栽谒拗髦惺褂玫倪@類標(biāo)記基因的例子包括氯霉素抗性基因、卡那霉素抗性基因、鏈霉素抗性基因、四環(huán)素抗性基因、甲氧芐啶抗性基因、紅霉素抗性基因等。
此外，如果通過將本發(fā)明的質(zhì)?；蚱洳糠峙c可以在其它細(xì)菌(如大腸桿菌)中自主復(fù)制的質(zhì)?；蚱浜衅鋸?fù)制控制區(qū)的部分相連接來將本發(fā)明的質(zhì)粒制備成可以在棒狀細(xì)菌和其它細(xì)菌(如大腸桿菌)中自主復(fù)制的穿梭載體的話，那么采用大腸桿菌可以進(jìn)行諸如質(zhì)粒的制備和含有靶基因的重組質(zhì)粒制備這樣的操作?？梢栽诖竽c桿菌中自主復(fù)制的質(zhì)粒的例子包括例如pUC19，pUC18，pBR322，pHSG299，pHSG298，pHSG399，pHSG398，RSF1010，pMW119，pMW118，pMW219，pMW218等。
盡管pYM1、pYM2、pYM3和pYM4本身的特征在于具有
圖1和圖2所示的限制圖，但并非必須要求本發(fā)明的質(zhì)粒具有這些限制圖，并且可以缺失任何的限制位點(diǎn)，而只要這種缺失不影響自主復(fù)制的能力。此外，本發(fā)明的質(zhì)?？梢园趐YM1、pYM2、pYM3和pYM4中所不含的限制位點(diǎn)。
以與常規(guī)公知的生產(chǎn)克隆載體、表達(dá)載體等相同的方式，可以構(gòu)建如上所述的質(zhì)粒衍生物。為了構(gòu)建出所述的衍生物，優(yōu)選測定pYM1、pYM2、pYM3和pYM4的核苷酸序列。可以通過如雙脫氧法這樣的公知方法來測定核苷酸序列。
為了將外源基因插入本發(fā)明的質(zhì)粒或其衍生物中，方便的作法是將其插入所述質(zhì)?；蚱溲苌锏南拗莆稽c(diǎn)中。作為這樣的一種限制位點(diǎn)，優(yōu)選以單一切割位點(diǎn)存在的位點(diǎn)。為了插入外源基因，可以用對兩者而言提供相同粘端的一種或多種限制酶(例如相同的限制酶)部分或完全地消化質(zhì)粒和外源基因的來源(如基因組DNA)，并且可以在適當(dāng)?shù)臈l件下使它們相連。還可以在平端使它們相連。
對于質(zhì)粒DNA的制備、DNA的消化和連接、轉(zhuǎn)化等來說，可以采用那些本領(lǐng)域普通技術(shù)人員眾所周知的方法。這樣的方法在Sambrook，J.，F(xiàn)ritsch，E.F.與Maniatis，T.的《分子克隆實(shí)驗(yàn)指南第2版》(冷泉港實(shí)驗(yàn)室出版社(1989))等文獻(xiàn)中進(jìn)行了描述。
根據(jù)本發(fā)明，如上所述提供了一種來源于熱產(chǎn)氨棒狀桿菌的新的質(zhì)粒。
實(shí)施例下面，參照以下實(shí)施例將更為詳細(xì)地解釋本發(fā)明。
實(shí)施例1來自熱產(chǎn)氨棒狀桿菌(FERM BP-1539，F(xiàn)ERM BP-1540，F(xiàn)ERM BP-1541.FERM BP-1542)的質(zhì)粒的分離和鑒定在CM2B液體培養(yǎng)基(細(xì)菌培養(yǎng)用胰化蛋白胨(Difco)1％，細(xì)菌培養(yǎng)用酵母提取物(Difco)1％，NaCl0.5％，生物素10μg/L)中培養(yǎng)熱產(chǎn)氨棒狀桿菌AJ12340(FERM BP-1539)、AJ12308(FERM BP-1540)、AJ12309(FERM BP-1541)和AJ12310(FERM BP-1542)12小時(shí)，并且通過堿法(《生物工程實(shí)驗(yàn)教材》由日本生物科學(xué)與生物工程協(xié)會(huì)編輯，第105頁，Baifukan，1992)獲得質(zhì)粒DNA的級分。當(dāng)通過瓊脂糖凝膠電泳分析這些級分時(shí)(Sambrook，J.，F(xiàn)ritsch，E.F.與Maniatis，T.《分子克隆實(shí)驗(yàn)指南 第2版》冷泉港實(shí)驗(yàn)室出版社(1989))，檢測所有情況下的DNA條帶，從而表明前面提到的菌株攜帶有質(zhì)粒。將由FERMBP-1540、FERM BP-1542和FERM BP-1539制得的質(zhì)粒分別命名為pYM1、pYM2和pYM3。將由FERM BP-1541制得的質(zhì)粒命名為pYM4。質(zhì)粒pYM1、pYM2和pYM3各自的長度約為4.4kb，而質(zhì)粒pYM4的長度約為6.0kb。
用限制酶BglⅡ、BamHⅠ、BstPⅠ、EcoRⅠ、HincⅡ、HindⅢ、KpnⅠ、NaeⅠ、NcoⅠ、NheⅠ、PmaCⅠ、SacⅠ、SacⅡ、SalⅠ、SmaⅠ、SphⅠ、TthlllⅠ和XbaⅠ(由Takara Co.生產(chǎn))酶切質(zhì)粒pYM1、pYM2、pYM3和pYM4，并通過瓊脂糖凝膠電泳測量所產(chǎn)生的DNA片段的長度。通過采用0.8％的瓊脂糖凝膠，在100V/cm和恒定的電壓下電泳幾個(gè)小時(shí)。作為分子量的標(biāo)記物，采用以限制酶HindⅢ消化的λ噬菌體DNA(Takara Shuzo)。在表1中顯示出針對pYM1、pYM2和pYM3獲得的結(jié)果。在表2中顯示出針對pYM4獲得的結(jié)果。在
圖1中顯示出pYM1、pYM2和pYM3的限制圖，在圖2中顯示出pYM4的限制圖，這些附圖都是基于以上的結(jié)果繪制的。
在SEQ ID NOS1、3、5和7中依次顯示出通過雙脫氧法測定的pYM1、pYM2、pYM3和pYM4的核苷酸序列的結(jié)果。
實(shí)施例2含有來源于糞鏈球菌的Km抗性基因的穿梭載體pYMFK的構(gòu)建作為在棒狀細(xì)菌中有效地復(fù)制pYM2所必須的區(qū)域，除了編碼rep的區(qū)域之外，還存在著位于rep上游的富含AT的區(qū)域以及位于rep下游的影響拷貝數(shù)的區(qū)域。
因此，為了獲得可以在棒狀細(xì)菌和大腸桿菌中復(fù)制、而不破壞pYM2的復(fù)制能力的穿梭載體，將能夠在大腸桿菌中自主復(fù)制的區(qū)域和選擇標(biāo)記插入位于pYM2的BstPⅠ位點(diǎn)附近的位點(diǎn)中。
首先，構(gòu)建具有糞鏈球菌的抗藥性基因的載體。由含有該基因的已知質(zhì)粒中通過PCR擴(kuò)增糞鏈球菌的卡那霉素抗性基因。已經(jīng)闡述了糞鏈球菌的卡那霉素抗性基因的核苷酸序列(Trieu-Cuot，P.與Courvalin，P.《基因》23(3)第331-341頁(1983))。在該序列的基礎(chǔ)上，合成了具有如SEQ ID NOS16和17所示核苷酸序列的引物，并且通過采用pDG783(Anne-Marie Guerout-Fleury等人《基因》167，第335-337頁(1995))作模板進(jìn)行PCR，以便擴(kuò)增出含有卡那霉素抗性基因及其啟動(dòng)子的DNA片段。
通過使用由Takara Shuzo有限公司生產(chǎn)的SUPREC02純化上述DNA片段，用限制酶HindⅢ和HincⅡ使其完全消化并形成平端。通過使用由Takara Shuzo有限公司生產(chǎn)的形成平端試劑盒來形成平端?；旌显揇NA片段和擴(kuò)增產(chǎn)物并使它們連接，所述的擴(kuò)增產(chǎn)物是通過采用具有如SEQ ID NOS18和19所示核苷酸序列的引物并以pHSG399(參見S.Takeshita等人《基因》61，第63-74頁(1987))作為模板進(jìn)行PCR以及純化并使PCR產(chǎn)物形成平端來獲得的。通過使用由Takara Shuzo有限公司生產(chǎn)的第2型DNA連接試劑盒進(jìn)行連接反應(yīng)。用連接的DNA轉(zhuǎn)化大腸桿菌JM109的感受態(tài)細(xì)胞(由Takara Shuzo有限公司生產(chǎn))，將其加入含有10μg/ml IPTG(異丙基-β-D-硫代吡喃半乳糖苷)、40μg/ml X-Gal(5-溴-4-氯-3-吲哚基-β-D-半乳糖苷)和25μg/ml卡那霉素的L培養(yǎng)基(10g/L細(xì)菌培養(yǎng)用胰化蛋白胨、5g/L細(xì)菌培養(yǎng)用酵母提取物、5g/LNaCl和15g/L瓊脂，pH7.2)中，并且培養(yǎng)過夜。然后，挑取形成的藍(lán)色菌落，并使其進(jìn)行單菌落的分離以獲得轉(zhuǎn)化體。
通過采用堿法(《生物工程實(shí)驗(yàn)教材》由日本生物科學(xué)與生物工程協(xié)會(huì)編輯，第105頁，Baifukan，1992)從轉(zhuǎn)化體中制備質(zhì)粒，并繪制出限制圖。把具有與圖6下部所示相同的限制圖的質(zhì)粒命名為pK1。該質(zhì)粒穩(wěn)定地?cái)y帶在大腸桿菌中，并賦予了宿主卡那霉素抗性。此外，由于它含有l(wèi)acZ’基因，因此它適合于用作克隆載體。
然后，采用從熱產(chǎn)氨棒狀桿菌AJ12310(FERM BP-1542)中提取出來的pYM2作模板(完整的pYM2的核苷酸序列示于SEQ ID NO9中)并采用在pYM2中靠近BstPⅠ位點(diǎn)的序列的基礎(chǔ)上制得的下列引物，通過Pyrobest-Taq(Takara Shuzo有限公司)擴(kuò)增含有復(fù)制起點(diǎn)的區(qū)域S15’-AACCAGGGGGAGGGCGCGAGGC-3’(SEQ ID NO10)S35’-TCT CGT AGG CTG CAT CCG AGG CGG GG-3’(SEQ ID NO11)。
反應(yīng)條件為94℃下5分鐘，然后是98℃下20秒鐘以及68℃下4分鐘的循環(huán)、該循環(huán)重復(fù)30次，以及72℃下4分鐘。反應(yīng)后，在4℃下儲存混合物。
通過使用由Amersham Pharmacia Biotech Co.生產(chǎn)的MicroSpin TMS-400 HR柱純化得到的擴(kuò)增片段，并通過采用由Takara Shuzo有限公司生產(chǎn)的DNA形成平端試劑盒使其形成平端，然后通過采用由TakaraShuzo有限公司生產(chǎn)的第2型DNA連接試劑盒使其與用HincⅡ處理的pK1連接。用連接的DNA轉(zhuǎn)化大腸桿菌JM109的感受態(tài)細(xì)胞(由TakaraShuzo生產(chǎn))以獲得轉(zhuǎn)化菌株。
采用堿法(《生物工程實(shí)驗(yàn)教材》由日本生物科學(xué)與生物工程協(xié)會(huì)編輯，第105頁，Baifukan，1992)從轉(zhuǎn)化菌株中制備質(zhì)粒，并繪制出質(zhì)粒的限制圖。把顯示出與圖3下部所示相同的限制圖的質(zhì)粒命名為pYMFK。pYMFK的大小約為7.0kb，它能夠在大腸桿菌和棒狀細(xì)菌中自主復(fù)制并且賦予了宿主Km抗性。
實(shí)施例3含有來源于Tn903的Km抗性基因的pYMK的構(gòu)建通過采用從熱產(chǎn)氨棒狀桿菌AJ12310(FERM BP-1542)中提取出來的pYM2作模板并采用下列引物，以與實(shí)施例2相同的方式擴(kuò)增含有復(fù)制起點(diǎn)的區(qū)域S1XbaⅠ5’-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG GC-3’(SEQ ID NO12)S3XbaⅠ5’-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG GCGGGG-3’(SEQ ID NO13)。
通過使用由Amersham Pharmacia Biotech Co.生產(chǎn)的MicroSpin TMS-400 HR柱純化得到的擴(kuò)增片段，并使用由Takara Shuzo有限公司生產(chǎn)的限制酶XbaⅠ進(jìn)行消化，然后通過采用由Takara Shuzo有限公司生產(chǎn)的第2型DNA連接試劑盒使其與用XbaⅠ完全消化pHSG299(TakaraShuzo有限公司)所得的片段連接。用連接的DNA轉(zhuǎn)化大腸桿菌JM109的感受態(tài)細(xì)胞(由Takara Shuzo生產(chǎn))以獲得轉(zhuǎn)化菌株。
采用堿法(《生物工程實(shí)驗(yàn)教材》由日本生物科學(xué)與生物工程協(xié)會(huì)編輯，第105頁，Baifukan，1992)從轉(zhuǎn)化菌株中制備質(zhì)粒，并繪制出質(zhì)粒的限制圖。把顯示出與圖4下部所示相同的限制圖的質(zhì)粒命名為pYMK。pYMK的大小約為7.0kb，它能夠在大腸桿菌和棒狀細(xì)菌中自主復(fù)制并且賦予了宿主Km抗性。
實(shí)施例4含有來源于Tn9的Cm抗性基因的穿梭載體pYMC的構(gòu)建通過采用從熱產(chǎn)氨棒狀桿菌AJ12310(FERM BP-1542)中提取出來的pYM2作模板并采用下列引物，以與實(shí)施例2相同的方式擴(kuò)增含有復(fù)制起點(diǎn)的區(qū)域S1XbaⅠ5’-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG GC-3’(SEQ ID NO14)S3XbaⅠ5’-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG GCGGGG-3’(SEQ ID NO15)。
通過使用由Amersham Pharmacia Biotech Co.生產(chǎn)的MicroSpin TMS-400 HR柱純化上述片段，并使用由Takara Shuzo有限公司生產(chǎn)的限制酶XbaⅠ進(jìn)行消化，然后通過采用由Takara Shuzo有限公司生產(chǎn)的第2型DNA連接試劑盒使其與用XbaⅠ處理pHSG399(Takara Shuzo有限公司)所得的片段連接。用連接的DNA轉(zhuǎn)化大腸桿菌JM109的感受態(tài)細(xì)胞(由Takara Shuzo生產(chǎn))以獲得轉(zhuǎn)化菌株。
采用堿法(《生物工程實(shí)驗(yàn)教材》由日本生物科學(xué)與生物工程協(xié)會(huì)編輯，第105頁，Baifukan，1992)從轉(zhuǎn)化菌株中制備質(zhì)粒，并繪制出質(zhì)粒的限制圖。把顯示出與圖5下部所示相同的限制圖的質(zhì)粒命名為pYMC。pYMC的大小約為6.6kb，它能夠在大腸桿菌和棒狀細(xì)菌中自主復(fù)制并且賦予了宿主Cm抗性。
序列表&#60110&#62Ajinomoto公司&#60120&#62在棒狀細(xì)菌中可自主復(fù)制的質(zhì)粒&#60130&#62&#60150&#62JP 11-228391&#60151&#621999-08-12&#60160&#6219&#60170&#62PatentIn 2.0版&#60210&#621&#60211&#621479&#60212&#62DNA&#60213&#62熱產(chǎn)氨棒狀桿菌&#60220&#62&#60221&#62CDS&#60222&#62(1)..(1476)&#60400&#621atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro85 90 95cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu115 120 125gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn
130 135 140ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr165 170 175acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576Thr Glu Glu Met Thr Arg Va1 Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190cgg ctg agc cgg tgg ccg ctg cat gtt tct gat gat ccg acc gcg tac 624Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr195 200 205tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210 215 220gag gta gcc cgc acg atg acc gga tca aaa aag ccc aga gag cac gct 720Glu Va1 Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Glu His Ala225 230 235 240cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260 265 270acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Va1 Leu275 280 285tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu385 390 395 400cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln405 410 415cga ctc gaa gct gca aac aag cga cgt caa gtc agc tgg aac aaa tac 1296Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr420 425 430gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu435 440 445gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser450 455 460cga gct acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys465 470 475 480tgc caa atc ctt agg ggg gct cac gcc gta gac aga taa 1479Cys Gln Ile Leu Arg Gly Ala His Ala Val Asp Arg485 490&#60210&#622&#60211&#62492&#60212&#62PRT&#60213&#62熱產(chǎn)氨棒狀桿菌&#60400&#622Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Va1 Ala Leu Thr His Pro85 90 95Gln Arg Ser Ala Vsl Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Va1 Tyr Ala Thr Leu115 120 125Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130135 140Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr165 170 175Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr195 200 205Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210 215 220Glu Vsl Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Glu His Ala225 230 235 240His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260 265 270Thr Ale Leu Glu Ale Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350Arg Gln Thr Met Ala Arg Arg Va1 Arg Ala Tyr Val Ala Lys Gly Gln355 360 365Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu385 390 395 400Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln405 410 415Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr420 425 430Ala Ser Thr Ash Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu435 440 445Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser450 455 460Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys465 470 475 480Cys Gln Ile Leu Arg Gly Ala His Ala Val Asp Arg485 490&#60210&#623&#60211&#621479&#60212&#62DNA&#60213&#62熱產(chǎn)氨棒狀桿菌&#60220&#62&#60221&#62CDS&#60222&#62(1)..(1476)&#60400&#623atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30cgg ggt tgg aat cac cag gac gcc gca gag ttc ctg gcc cac ctg gat 144Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro85 90 95cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu115 120 125gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130 135 140ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr165 170 175acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190cgg ctg agc cgg tgg ccg ctg cat gtt ttt gat gat ccg acc gcg tac 624Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr195 200 205tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210 215 220gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala225 230 235 240cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260 265 270acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu385 390 395 400cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln
405 410 415cga ctc gaa gct gca aac aag cga cgt caa gtc agc tgg aac aaa tac 1296Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr420 425 430gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu435 440 445gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser450455 460cga gct acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys465 470 475480tgc caa atc ctt agg ggg gct cac gcc gta cac aga taa 1479Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg485 490&#60210&#624&#60211&#62492&#60212&#62PRT&#60213&#62熱產(chǎn)氨棒狀桿菌&#60400&#624Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro85 90 95Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu115 120 125Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130 135 140Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145150 155 160Ale Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ale Ala Thr165 170 175Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr195 200205Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210 215 220Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala226 230 235 240His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro
260 265 270Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285Trp Ale Ala Glu Gly Arg Ale Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300Ala Leu Thr Va1 Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu385 390 395 400Arg Trp Lys Thr Asp Pro ASh Gly Lys Tyr Ala Gln Glu Ash Arg Gln405 410 415Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr420 425 430Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu435 440 445Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser450 455 460Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys465 470 475 480Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg485 490&#60210&#625&#60211&#621479&#60212&#62DNA&#60213&#62熱產(chǎn)氨棒狀桿菌&#60220&#62&#60221&#62CDS&#60222&#62(1)..(1476)&#60400&#625atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro85 90 95cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110gcc ggc ggg aac atc gag cac ctt cac ccg cag gta tac gcc acc ttg 384Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu115 120 125gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130 135 140ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr165 170 175acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190cgg ctg agc cgg tgg ccg ctg cat gtt tct gat gat ccg acc gcg tac 624Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr195 200 205tcc tgg cac tgc cag cac aac cga gtc gat acg ctt gat gag ctg atg 672Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Asp Glu Leu Met210 215 220gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala225 230 235 240cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260 265 270acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu385 390 395 400cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln405 410415cga ctc gaa gct gca sac aag cga cgt caa gtc agc tgg aac aaa tac 1296Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr420 425 430gcg agc acg aat tct ggc tac ggt ttc cea cac eta tgg gcc agc ttg 1344Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Van Trp Ala Ser Leu435 440 445gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser450 455 460cga gct acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys465 470 475 480tgc caa atc ctt agg ggg gct cac gcc gta cac aga taa 1479Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg485 490&#60210&#626&#60211&#62492&#60212&#62PRT&#60213&#62熱產(chǎn)氨棒狀桿菌&#60400&#626Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro85 90 95Gln Arg Ser Ala Val Leu Va1 Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Va1 Tyr Ala Thr Leu115 120 125Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130 135 140Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr165 170 175Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr195 200 205Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Asp Glu Leu Met210 215 220Glu Va1 Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala225 230 235240His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260265 270Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu385 390 395 400Arg Trp Lys Thr Asp Pro ASh Gly Lys Tyr Ala Gln Glu Asn Arg Gln405 410 415Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr420 425 430Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu435 440 445Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser450 455 460Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys465 470 475 480Cys Gln Ile Leu Arg Gly Als His Ala Val His Arg485 490&#60210&#627&#60211&#621377&#60212&#62DNA&#60213&#62熱產(chǎn)氨棒狀桿菌&#60220&#62&#60221&#62CDS&#60222&#62(1)..(1374)&#60400&#627atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15tac tcc aca gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp
50 55 60gtc aaa gac ccg gag acc gga gaa acc cgc cct aag ctg cac aga gtc 240Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80ggc acc cgg tcg ctt tcc cga tgc cag tac gtc gcg ctg acc cac ccg 288Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro85 90 95cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110gcc ggc ggg aac atc gag cac ctt cac ccg cag gtc tac gcc acc ttg 384Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu115 120 125gag cgc tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130 135 140ccg ttg tca gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160gcc gcc gag ggc acc acc age ccg aac acc cgc ctg cta gcg gcc acc 528Ala Ala Glu Gly Thr Thr Ser Pro Asn Thr Arg Leu Leu Ala Ala Thr165 170 175acc gag gaa atg acc cgt atg ttc ggc gct gac cag gca ttt tcc cac 576Thr Glu Glu Met Thr Arg Met Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190cgg ctg agc cgg tgg ccg ctg cat gta tct gat gat ccg acc gcg tac 624Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr195 200 205tcc tgg cac tgc cag cac aac cga gtc gat acg ctt gct gag ctg atg 672Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Ala Glu Leu Met210 215 220gag gta gcc cgc acg atg acc gga tca aaa aag cca gat agc act gct 720Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Asp Ser Thr Ala225 230 235 240cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg agg aaa 768His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255gcc acc gca gaa gcc aaa gcg ctt gct gcc tta gac gcc acg ctg cct 816Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260 265 270acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ctg 864Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285tgg gca gca gag ggg cgt gca gcc cgt gat gag acg gcg ttt cgc cat 912Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300gcg ttg acc gtg ggg tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320gac acc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008Asp Thr Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335gcg gtg ggg gct gat ggg cgt gag ccg gat ctg cct gcc atg cgt gat 1056Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350cgt cag acg ttg gcc cgt cgt gtg cgc gcc tac gtc gct aaa ggc cag 1104Arg Gln Thr Leu Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365ccc acg gtg agc gcc agg agc aca cag acc cag agc agc cgg ggc agg 1152Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380aaa gcc ctg gcc acc atg gga cgc aga ggc gca gcc acc tcg aat gca 1200Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Ala Ala Thr Ser Asn Ala385 390 395 400cgc agg tgg gca gac cca gaa agc gat tac gcc cgc caa act cgg gag 1248Arg Arg Trp Ala Asp Pro Glu Ser Asp Tyr Ala Arg Gln Thr Arg Glu405 410 415cgt tta gcc cga gca atg agc ttc gta cat tca gca cag acg age aca 1296Arg Leu Ala Arg Ala Met Ser Phe Val His Ser Ala Gln Thr Arg Thr420 425 430agg gcc gga tcc tgg cct acg ttt ccg agt gca agc gcc acg gtt acg 1344Arg Ala Gly Ser Trp Pro Thr Phe Pro Ser Ala Ser Ala Thr Val Thr435 440 445acc cca cga gca aag aag tcg caa cgg agc tag 1377Thr Pro Arg Ala Lys Lys Ser Gln Arg Ser450 455&#60210&#628&#60211&#62458&#60212&#62PRT&#60213&#62熱產(chǎn)氨棒狀桿菌&#60400&#628Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Va1 Ala Leu Thr His Pro85 90 95Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu115 120 125Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130 135 140Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160Ala Ala Glu Gly Thr Thr Ser Pro Asn Thr Arg Leu Leu Ala Ala Thr165 170 175Thr Glu Glu Met Thr Arg Met Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr195 200 205Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Ala Glu Leu Met210 215 220Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Asp Ser Thr Ala225 230 235 240His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260 265 270Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320Asp Thr Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350Arg Gln Thr Leu Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Ala Ala Thr Ser Asn Ala385 390 395 400Arg Arg Trp Ala Asp Pro Glu Ser Asp Tyr Ala Arg Gln Thr Arg Glu405 410 415Arg Leu Ala Arg Ala Met Ser Phe Val His Ser Ala Gln Thr Arg Thr420 425 430Arg Ala Gly Ser Trp Pro Thr Phe Pro Ser Ala Ser Ala Thr Val Thr435 440 445Thr Pro Arg Ale Lys Lys Ser Gln Arg Ser450 455&#60210&#629&#60211&#624369&#60212&#62DNA&#60213&#62熱產(chǎn)氨棒狀桿菌&#60220&#62&#60221&#62CDS&#60222&#62(1)..(1476)&#60400&#629atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat48Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn1 5 10 15tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc96Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser20 25 30cgg ggt tgg aat cac cag gac gcc gca gag ttc ctg gcc cac ctg gat 144Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp35 40 45cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp50 55 60gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val65 70 75 80ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro85 90 95cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln100 105 110gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu115 120 125gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn130 135 140ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe145 150 155 160gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr165 170175acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His180 185 190cgg ctg agc cgg tgg ccg ctg cat gtt ttt gat gat ccg acc gcg tac 624Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr195 200 205tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210215 220gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala225 230 235 240cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys245 250 255gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro260 265 270acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu275 280 285tgg gca gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His290 295 300gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaa cgc ctg aaa 960Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys305 310 315 320gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln325 330 335gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp340 345 350cgt cag acg atg gcc cgc cgt gtg cgc gcc tac gtc gcc aaa ggc cag 1104Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln355 360 365ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg370 375 380aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu385 390 395 400cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln405 410 415cga ctc gaa gct gca sac aag cga cgt caa gtc agc tgg aac aaa tac 1296Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr420 425 430gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu435 440 445gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser450 455 460cga gct acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys465 470 475 480tgc caa atc ctt agg ggg gct cac gcc gta cac aga taacggttcc cacccc 1492Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg485 490gtaggggtag cgcttggtcc ctgaagctcc ggctcccatc cctcctcagc actccctccc 1552cgaggggggg gctcacgccg tagacagata acggttccca ccccgtaggg gtagcgcttg 1612gtccctgaag ctctcacttc tggctcccct cctggccctc cttgagtgcc cacccataaa 1672tgcgaaatgc cgtcagcaga caacggttcc cacccctggg gtcctcacaa caggctgcat 1732cagggctctc gacgcttgct ggcttcatcc atcaactgct gggtgatctc ctcgaacgca 1792tccttgatcg cgagttcctc gaaatcagcg gcagcttgct cccagtggat ccgttccagc 1852tccggcagcg ccgcccacag cgccatgcgc aagtaggttc cacgtgatcg cttagtgcgc 1912tcggcgagcg cgtccaggcg ctcgatcagc tccggttcca ggcgcacgga gaccacgggg 1972ccgcgtccgg cggggttctt ctggttggtg gccatgagaa atttcctctc gcttcggtag 2032ttgtaaacaa tgtttacacc gtgtcgggga gaggggtttt tatttttctc ccgggcactt 2092tcgagacggg tcatgccgta agcgaggcgc gtggccacac cgcactcggc gacgcaggtg 2152tcacttgctc cccgactgtc ggccgggaag ggggcgcgca gagcgtccag gagcgccgta 2212gagcgcctgg gacggttcgt gtggggactt ggtcgcccca cggggcttta atcgcttaaa 2272aacgcgcaca gcgcatttct tgccacgggc tagcgcgtga ccgctgcgcg ctcacttgct 2332caggaagaaa atcattcctc gcctaaagcg cttcgcgcgc tcgccctctc cgagggggaa 2392aactaaccac acacctcatg cactaaagtg ctgatttgca ggtcagcgcg ttttagcgtg 2452caaaaatagt gcggaaaacg gcgaaaatgg gggcgcgaca atcccctcag tggctcccca 2512aaattcacct attcacatct gctactggct gacttctttc ccgacaaggg gccctgtgag 2572ggcgcaggtt gagccacttt tacgtcccgg agatcccttt agggcgtatt cgaggtgtgc 2632tcagtgaccc gcttcggcgg ggtgggagta gccaaaagtc cgacattttt aacgaacgtt 2692cgttaaaatg ggggcatgac tcagggacct ttgacctcag aaaccggcgc aatcctgaat 2752gatcttggcg cagcagaccc tctcgatgtg gctgtccggg cacgggagag tgcgcatgtt 2812ctctctcaag tcgtggagtt tttagagcag atcggccggt ctggggatag cgatttagac 2872gcggtgtatg agcgtgattg gcagctcgat gcagacacgt tgaccttcat tgcccaggcg 2932ttggaggggt tggcggacca ggccgaggcg aaggatgccg tgaacgaatg acggatatgt 2992gtgcccaatg cggtggggaa atcccgcccc ggcctgaccc ccgcggacgc agggcgaagt 3052attgctcgga tgcttgtcgg gcggcagcga gccgcgaacg cgcgcgccag cgccacgccc 3112aggaggtcga agccgcgcgt ctccaggccg cactcgatct gaaaaccccg caggagaccc 3172tggcagaggt agtccaggag cttcaggcca ccacccggat tatccgtgat cgaggggacg 3232tgccagcgtc gctgcgtccg ctggttaatg ctgcatccga actggtcaac gcagcgcaac 3292cggttgagga atctaagtca ttccccaacc ggcgagtgcg tcgtgcagtt aaacgaaagt 3352ttgcgataag cgggtgatgt aactgatgga gatttttacc tgggggtgtc tccagcgagg 3412tggccaagtc cgattgtgtt gaggattacc ccaaacgtgc ggggattatt caaaatccac 3472tgtccaaccg cttttccggt taccccgcct ccgatgcagc ctacgagaat agagcccatg 3532accattgcat tgtggctata tcccgcattt ggatccagcg ccgagaaact ggtgtaggca 3592ccagcagcgc agcctgcaat gcgagcgcca atgataacca gggggagggc gcgaggcatt 3652actcgatttt catctgtggt ctgtcgctga atcgaagcag tgatggcttc ttcaaatgct 3712tcagggtttg acgtggggtc cgagactgtt gacgcagctt cctgcactgc cttgatgagg 3772acatcttcag ggatggaatc attgaacatt cctcccagct cagaagtggt ttgaacgtta 3832gccgaaggga catgcacatc gggggaagcc tgggcggctg gagcaattaa agacagcgac 3892agtgaagcaa cgagagccgt tacagtggca cgagttttta aatacatgag gcgaacttaa 3952caaaccattg ataggttgtc gtgcggtaaa gataagaaaa ggataaagat atgaaaacgt 4012tatttatgaa tctcttaggt gccgcgcttg taggagcggt aatcatggtc ttgacatggt 4072tatttattga ttttgatgca cctggagcat ggctcggatt ctttattatc accaccatca 4132gtgattgctg ctttagaagt catccacgga ctttgggaaa aacggcaggg atcttccact 4192gacaatgatt gataaaacct ggttgaacgg aatacaaaac gcgcaaaata accaggcagt 4252taaaagaaaa accagataag ctgcaccaat acttgaaaaa tgttgaacgc cccgacagct 4312gtaactgtcg aggcgtcggc taacccccag tcatcagctg ggagaaagca ctcaaaa4369&#60210&#6210&#60211&#6222&#60212&#62DNA&#60213&#62人工序列&#60223&#62人工序列的描述用于擴(kuò)增pYM2的復(fù)制起點(diǎn)的引物&#60400&#6210aaccaggggg agggcgcgag gc 22&#60210&#6211&#60211&#6226&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增pYM2的復(fù)制起點(diǎn)的引物&#60400&#6211tctcgtaggc tgcatccgag gcgggg 26&#60210&#6212&#60211&#6232&#60212&#62dna&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增pYM2的復(fù)制起點(diǎn)的引物&#60400&#6212gctctagagc aaccaggggg agggcgcgag gc 32&#60210&#623&#60211&#6236&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增pYM2的復(fù)制起點(diǎn)的引物&#60400&#6213gctctagagc tctcgtaggctgcatcggag gcgggg36&#60210&#6214&#60211&#6232&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增pW2的復(fù)制起點(diǎn)的引物&#60400&#6214gctctagagc aaccaggggg agggcgcgag gc 32&#60210&#6215&#60211&#6236&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增pYM2的復(fù)制起點(diǎn)的引物&#60400&#6215gctctagagc tctcgtaggc tgcatcggag gcgggg36&#60210&#6216&#60211&#6232&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增糞鏈球菌的卡那霉素抗性基因的引物&#60400&#6216cccgttaact gcttgaaacc caggacaata ac32&#60210&#6217&#60211&#6230&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增糞鏈球菌的卡那霉素抗性基因的引物&#60400&#6217cccgttaaca tgtacttcag aaaagattag 30&#60200&#6218&#60211&#6226&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增大腸桿菌克隆載體pHSG399的引物&#60400&#6218gatatctacg tgccgatcaa cgtctc26&#60210&#6219&#60211&#6225&#60212&#62DNA&#60213&#62人工序列&#60220&#62&#60223&#62人工序列的描述用于擴(kuò)增大腸桿菌克隆載體pHSG399的引物&#60400&#6219aggccttttt ttaaggcagt tattg 2權(quán)利要求
1．一種可以從熱產(chǎn)氨棒狀桿菌(Corynebacteriumthermoaminogenes)中分離的質(zhì)?；蚱溲苌铮撡|(zhì)粒包含編碼Rep蛋白的基因并且大小約為4.4kb或約為6kb，所述的Rep蛋白具有SEQ IDNO2中所示的氨基酸序列或具有與SEQ ID NO2中所示的氨基酸序列90％或更高同源性的氨基酸序列。
2．根據(jù)權(quán)利要求1的質(zhì)?；蚱溲苌?，它可以從熱產(chǎn)氨棒狀桿菌AJ12340(FERM BP-1539)、AJ12308(FERM BP-1540)或AJ12310(FERMBP-1542)中分離出來，它的大小約為4.4kb并且由
圖1中所示的限制圖表示。
3．根據(jù)權(quán)利要求1的質(zhì)粒或其衍生物，它可以從熱產(chǎn)氨棒狀桿菌AJ12309(FERM BP-1541)中分離出來，它的大小約為6kb并且由圖2中所示的限制圖表示。
4．根據(jù)權(quán)利要求1的質(zhì)?；蚱溲苌铮幋aRep蛋白的基因，所述的Rep蛋白具有SEQ ID NO2、4或6中所示的氨基酸序列。
5．根據(jù)權(quán)利要求1的質(zhì)?；蚱溲苌铮幋aRep蛋白的基因，所述的Rep蛋白具有SEQ ID NO8中所示的氨基酸序列。
全文摘要
一種可以從熱產(chǎn)氨棒狀桿菌(Corynebacterium thermoaminogenes)中分離的質(zhì)粒或其衍生物,該質(zhì)粒包含編碼Rep蛋白的基因并且大小約為4.4kb或約為6kb,所述的Rep蛋白具有SEQ ID NO:2中所示的氨基酸序列或具有與SEQ ID NO:2中所示的氨基酸序列90%或更高同源性的氨基酸序列。
文檔編號C12N1/21GK1288060SQ00130669
公開日2001年3月21日 申請日期2000年8月12日 優(yōu)先權(quán)日1999年8月12日
發(fā)明者松崎友美, 木村英一郎, 中松亙, 倉橋修, 河原義雄, 杉本慎一 申請人:味之素株式會(huì)社