專利名稱：抗隱孢子蟲重組免疫毒素蛋白及其制備工藝和醫(yī)用用途的制作方法
技術(shù)領(lǐng)域：
本發(fā)明公開一種抗隱孢子蟲重組免疫毒素蛋白及其制備工藝和醫(yī)用用途，涉及預(yù)防和治療隱孢子蟲病的藥物制備技術(shù)領(lǐng)域。
背景技術(shù)：
隱孢子蟲病(Cryptosporidiosis)是一種重要的人獸共患寄生蟲病，該病具有以下特點(diǎn)該蟲宿主范圍極為廣泛、感染率高、危害嚴(yán)重，隱孢子蟲病可引起人和牛的嚴(yán)重腹瀉，是艾滋病人的主要致死因素之一。犢牛隱孢子蟲病死亡率高達(dá)53.5％，奶牛感染隱孢子蟲后產(chǎn)奶量可降低3.2kg/d，耐過動物可成為帶蟲者和傳染源，而且隱孢子蟲可造成T細(xì)胞數(shù)量的急劇減少使機(jī)體免疫力降低。90％以上的隱孢子蟲病人有腹瀉癥狀，多呈水樣便，并伴有腹痛、腹脹、嘔吐、乏力和發(fā)熱等。免疫功能缺陷者感染隱孢子蟲后可引起嚴(yán)重的腹瀉并危及生命。因此本病是今后醫(yī)學(xué)和獸醫(yī)界防治的重要寄生蟲病。
迄今已有200余種藥物包括所有抗生素、抗球蟲藥及磺胺類藥等，被試用于治療人和動物隱孢子蟲病，但尚無一種藥物對隱孢子蟲有真正殺滅作用。長期以來人們期望的一種僅殺傷有害細(xì)胞而不損傷正常細(xì)胞的導(dǎo)向藥物在腫瘤的治療中發(fā)揮了重要作用。同時(shí)也顯示出它在解決迄今缺乏有效治療藥物的感染性疾病方面的具大潛力，已成為一個活躍的研究領(lǐng)域。

發(fā)明內(nèi)容
本發(fā)明提供一種抗隱孢子蟲重組免疫毒素蛋白及其制備工藝，可適用于工業(yè)化生產(chǎn)。
本發(fā)明還提供了抗隱孢子蟲重組免疫毒素蛋白在預(yù)防和治療人和動物隱孢子蟲病的醫(yī)用用途。
抗隱孢子蟲重組免疫毒素蛋白的基因序列為兩個，其中，第一個基因序列為CAG GTG AAG CTG CAG GAG TCA GGA CCT GGC CTG GTG GCA CCC TCA 45Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser1 5 10 15CAG AGC CTG TCC ATC ACA TGC ACT GTC TCT GGG TTC TCA TTA TCC 90Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser16 20 25 30AGA TAT AGT GTA CAC TGG GTT CGC CAG CCT CCA GGA AAG GGT CTG 135Arg Tyr Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu31 35 40 45GAG TGG CTG GGA ATG ATA TGG GGT GGT GGA AGC ACA GAC TAT AAT 180Glu Trp Leu Gly Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn46 50 55 60TCA GCT CTC AAA TCC AGA CTG AGC ATC AGC AAG GAC AAC TCC AAG 225Ser Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys61 65 70 75AGC CAA GTT TTC TTA AAA ATG AAC AGT CTG CAA ACT GAT GAC ACA 270Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr76 80 85 90GCC ATG TAC TAC TGT GCC AGA AAC CGG TAT GGT AAT GCT ATG GAC 315Ala Met Tyr Tyr Cys Ala Arg Asn Arg Tyr Gly Asn Ala Met Asp91 95 100 105TAC TGG GGC CAA GGC ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC 360Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly106 110 115 120GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG GAC ATC GAG 405Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu121 125 130 135CTC ACT CAG TCT CCA GCC TCC CTA TCT GTA TCT GTG GGA GAA ACT 450Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly Glu Thr136 140 145 150GTC ACC ATC ACA TGT CGA GCA AGT GAG AAT ATT TAC AGT AAT TTA 495Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Tyr Ser Asn Leu151 155 160 165GCA TGG TAT CAG CAG AAA CAG GGA AAA TCT CCT CAG CTC CTG GTC 540Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val166 170 175 180
TAT GCT GCA ACA AAC TTA GCA GAT GGT GTG CCA TCA AGG TTC AGT 585Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser181 185 190 195GGC AGT GGA TCA GGC ACA CAG TAT TCC CTC AAG ATC AAC AGC CTG 630Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu196 200 205 210CAG TCT GAA GAT TTT GGG AGT TAT TAC TGT CAA CAT TTT TGG GGT 675Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly211 215 220 225ACT CCG TAC ACG TTC GGA GGG GGG AGC AAG CTG GAG CTG AAA CGG 720Thr Pro Tyr Thr Phe Gly Gly Gly Ser Lys Leu Glu Leu Lys Arg226 230 235 240CAT ATG GCC GAA GAG GGC GGC AGC CTG GCC GCG CTG ACC GCG CAC 765His Met Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His241 245 250 255CAG GCT TGC CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT CGC CAG 810Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln256 260 265 270CCG CGC GGC TGG GAA CAA CTG GAG CAG TGC GGC TAT CCG GTG CAG 855Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Thr Pro Val Gln271 275 280 285CGG CTG GTC GCC CTC TAC CTG GCG GCG CGG CTG TCG TGG AAC CAG 900Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln286 290 295 300GTC GAC CAG GTG ATC CGC AAC GCC CTG GCC AGC CCC GGC AGC GGC 945Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly301 305 310 315GGC GAC CTG GGC GAA GCG ATC CGC GAG CAG CCG GAG CAG GCC CTG 990Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Leu316 320 325 330GCC CTG ACC CTG GCC GCC GCC GAG AGC GAG CGC TTC GTC CGG CAG 1035Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln331 335 340 345GGC ACC GGC AAC GAC GAG GCC GGC GCG GCC AAC GCC GAC GTG GTG 1080Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val346 350 355 360AGC CTG ACC TGC CCG GTC GCC GCC GGT GAA TGC GCG GGC CCG GCG 1125Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala361 365 370 375GAC AGC GGC GAC GCC CTG CTG GAG CGC AAC TAT CCC ACT GGC GCG 1170Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala376 380 385 390GAG TTC CTC GGC GAC GGC GGC GAC GTC AGC TTC AGC ACC CGC GGC 1215Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly
391 395 400 405ACG CAG AAC TGG ACG GTG GAG CGG CTG CTC CAG GCG CAC CGC CAA 1260Thr Glu Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln406 410 415 420CTG GAG GAG CGC GGC TAT GTG TTC GTC GGC TAC CAC GGC ACC TTC 1305Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe421 425 430 435CTC GAA GCG GCG CAA AGC ATC GTC TTC GGC GGG GTG CGC GCG CGC 1350Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg436 440 445 450AGC CAG GAC CTC GAC GCG ATC TGG CGC GGT TTC TAT ATC GCC GGC 1395Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly451 455 460 465GAT CCG GCG CTG GCC TAC GGC TAC GCC CAG GAC CAG GAA CCC GAC 1440Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp466 470 475 480GCA CGC GGC CGG ATC CGC AAC GGT GCC CTG CTG CGG GTC TAT GTG 1485Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val481 485 490 495CCG CGC TCG AGC CTG CCG GGC TTC TAC CGC ACC AGC CTG ACC CTG 1530Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu496 500 505 510GCC GCG CCG GAG GCG GCG GGC GAG GTC GAA CGG CTG ATC GGC CAT 1575Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His511 515 520 525CCG CTG CCG CTG CGC CTG GAC GCC ATC ACC GGC CCC GAG GAG GAA 1620Pro leu Pro Leu Arg leu Asp Ala Ile Thr Gly Pro Glu Glu Glu526 530 535 540GGC GGG CGC CTG GAG ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC 1665Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg541 545 550 555ACC GTG GTG ATT CCC TCG GCG ATC CCC ACC GAC CCG CGC AAC GTC 1710Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val556 560 565 570GGC GGC GAC CTC GAC CCG TCC AGC ATC CCC GAC AAG GAA CAG GCG 1755Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala571 575 580 585ATC AGC GCC CTG CCG GAC TAC GCC AGC CAG CCC GGC AAA CCG CCG 1800Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro586 590 595 600CGC GAG GAC CTG AAG TAA CTG CCG CGA CCG GCC GGC TCC CTT CGC 1845Arg Glu Asp Leu Lys ***601 605AGG AGC CGG CCT TCT CGG GGC CTG GCC ATA CAT CAG GTT TTC CTG 1890
ATG CCA GCC CAA TCG AAT ATG AAT TC第二個基因序列為CAG GTG AAG CTG CAG GAG TCT GGG GGA GGC TTA GTG AAG CCT GGA 45Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly1 5 10 15GGG TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA TTC ACT TTC AGT 90Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Ala Phe Thr Phe Ser16 20 25 30AGC TAT GCC ATG TCT TGG GTT CGC CAG ACT CCG GAG AAG AGG CTG 135Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu31 35 40 45GAG TGG GTC GCA ACC ATT AGT GGT GGT GGT AGT TAC ACC TAC TAT 180Glu Trp Val Ala Thr Leu Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr46 50 55 60CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC AGA GAC AAT GCC 225Pro Asp Ser Val Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala61 65 70 75AAG AAC ACC CTG TAC CTG CAA ATG AGC AGT CTG AGG TCT GAG GAC 270Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp76 80 85 90ACG GCC ATG TAT TAC TGT GCA AGA CGG GAG GGC TGG TTT GCT TAC 305Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Gly Trp Phe Ala Tyr91 95 100 105TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT 350Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly106 110 115 120TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG TAC ATC GAG CTC 395Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Ile Glu Leu121 125 130 135ACT CAG TCT CCA GCC TCC CTA TCT GCA TCT GTG GGA GAA ACT GTC 450Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr Val136 140 145 150ACC ATC ACA TGT CGA GCA AGT GGG AAT ATT CAC AAT TAT TTA GCA 495Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala151 155 160 165TGG TAT CAG CAG AAA CAG GGA AAA TCT CCT CAG CTC CTG GTC TAT 540Trp Tyr Gln Gln Lys Gly Gln Lys Ser Pro Gln Leu Leu Val Tyr166 170 175 180AAT GCA AAA ACC TTA GCA GAT GGT GTG CCA TCA AGG TTC AGT GGC 585Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly181 185 190 195AGT GGA TCA GGA ACA CAA TAT TCT CTC AAG ATC AAC AGC CTG CAG 630
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln196 200 205 210CCT GAA GAT TTT GGG AGT TAT TAC TGT CAA CAT TTT TGG AGT ACT 675Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Tyr211 215 220 225CCT CCC ACG TTC GGA GGG GGG ACC AAG CTG GAT CTG AAA CCG CAT 720Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Asp Leu Lys Pro His226 230 235 240ATG GCC GAA GAG GGC GGC AGC CTG GCC GCG CTG ACC GCG CAC CAG 765Met Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln241 245 250 255GCT TGC CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT CGC CAG CCG 810Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro256 260 265 270CGC GGC TGG GAA CAA CTG GAG CAG TGC GGC TAT CCG GTG CAG CGG 855Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Thr Pro Val Gln Arg271 275 280 285CTG GTC GCC CTC TAC CTG GCG GCG CGG CTG TCG TGG AAC CAG GTC 900Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val286 290 295 300GAC CAG GTG ATC CGC AAC GCC CTG GCC AGC CCC GGC AGC GGC GGC 945Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly301 305 310 315GAC CTG GGC GAA GCG ATC CGC GAG CAG CCG GAG CAG GCC CTG GCC 990Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Leu Ala316 320 325 330CTG ACC CTG GCC GCC GCC GAG AGC GAG CGC TTC GTC CGG CAG GGC 1035Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly331 335 340 345ACC GGC AAC GAC GAG GCC GGC GCG GCC AAC GCC GAC GTG GTG AGC 1080Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser346 350 355 360CTG ACC TGC CCG GTC GCC GCC GGT GAA TGC GCG GGC CCG GCG GAC 1125Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp361 365 370 375AGC GGC GAC GCC CTG CTG GAG CGC AAC TAT CCC ACT GGC GCG GAG 1170Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu376 380 385 390TTC CTC GGC GAC GGC GGC GAC GTC AGC TTC AGC ACC CGC GGC ACG 1215Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr391 395 400 405CAG AAC TGG ACG GTG GAG CGG CTG CTC CAG GCG CAC CGC CAA CTG 1260Glu Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu406 410 415 420
GAG GAG CGC GGC TAT GTG TTC GTC GGC TAC CAC GGC ACC TTC CTC 1305Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu421 425 430 435GAA GCG GCG CAA AGC ATC GTC TTC GGC GGG GTG CGC GCG CGC AGC 1350Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser436 440 445 450CAG GAC CTC GAC GCG ATC TGG CGC GGT TTC TAT ATC GCC GGC GAT 1395Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp451 455 460 465CCG GCG CTG GCC TAC GGC TAC GCC CAG GAC CAG GAA CCC GAC GCA 1440Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala466 470 475 480CGC GGC CGG ATC CGC AAC GGT GCC CTG CTG CGG GTC TAT GTG CCG 1485Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro481 485 490 495CGC TCG AGC CTG CCG GGC TTC TAC CGC ACC AGC CTG ACC CTG GCC 1530Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala496 500 505 510GCG CCG GAG GCG GCG GGC GAG GTC GAA CGG CTG ATC GGC CAT CCG 1575Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro511 515 520 525CTG CCG CTG CGC CTG GAC GCC ATC ACC GGC CCC GAG GAG GAA GGC 1620leu Pro Leu Arg leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly526 530 535 540GGG CGC CTG GAG ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC ACC 1665Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr541 545 550 555GTG GTG ATT CCC TCG GCG ATC CCC ACC GAC CCG CGC AAC GTC GGC 1710Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly556 560 565 570GGC GAC CTC GAC CCG TCC AGC ATC CCC GAC AAG GAA CAG GCG ATC 1755Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile571 575 580 585AGC GCC CTG CCG GAC TAC GCC AGC CAG CCC GGC AAA CCG CCG CGC 1800Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg586 590 595 600GAG GAC CTG AAG TAA CTG CCG CGA CCG GCC GGC TCC CTT CGC AGG 1845Glu Asp Leu Lys ***601 605AGC CGG CCT TCT CGG GGC CTG GCC ATA CAT CAG GTT TTC CTG ATG 1890CCA GCC CAA TCG AAT ATG AAT TC
本發(fā)明抗隱孢子蟲重組免疫毒素蛋白制備工藝包括以下步驟利用單克隆抗體技術(shù)和基因重組技術(shù)，從分泌抗微小隱孢子蟲(Cryptosporidium parvum)子孢子表膜抗體的雜交瘤細(xì)胞株中克隆其可變區(qū)基因，再連接成單鏈抗體基因，然后將其插入原核表達(dá)載體的相應(yīng)位點(diǎn)上，構(gòu)建了重組原核表達(dá)質(zhì)粒，再分別采用酶切和PCR方法將去除受體結(jié)合區(qū)的細(xì)菌外毒素插入重組原核質(zhì)粒中單鏈抗體基因的下游位點(diǎn)，即利用單鏈抗體基因代替細(xì)菌外毒素的受體結(jié)合區(qū)，構(gòu)建重組表達(dá)質(zhì)粒。將上述構(gòu)建的重組表達(dá)質(zhì)粒分別轉(zhuǎn)化受體菌，經(jīng)IPTG誘導(dǎo)，SDS-PAGE分析表達(dá)產(chǎn)物，后對表達(dá)產(chǎn)物包涵體進(jìn)行純化和復(fù)性，并對其進(jìn)行體外抗蟲活性測定和模型動物隱孢子蟲病治療。
通過以下試驗(yàn)例證明抗隱孢子蟲重組免疫毒素蛋白對于對隱孢子蟲疾病的預(yù)防和治療作用實(shí)驗(yàn)例1.
60只小鼠每只經(jīng)口接種1×106個隱孢子蟲后，隨機(jī)將其分為六組，一組至五組為治療組，六組為對照組。治療組每日經(jīng)肛門分別灌入小腸不同劑量(0.5，1，1.5，2，2.5g)的重組毒素，連續(xù)3天，對照組以生理鹽水代替重組毒素。7天后，取1克糞便計(jì)算每克糞便中的卵囊數(shù)。結(jié)果顯示，治療組卵囊數(shù)明顯少于對照組(p＜0.05)。其中治療的最佳劑量為1g/只鼠。
實(shí)施例2.
60只小鼠每只經(jīng)口接種1×106個隱孢子蟲后，隨機(jī)分為六組，一組至五組為治療組，六組為對照組。治療組每日經(jīng)肛門分別灌入小腸不同劑量(0.5，1，1.5，2，2.5g)的重組毒素，連續(xù)3天，對照組以生理鹽水代替重組毒素。7天后，取1克糞便計(jì)算每克糞便中的卵囊數(shù)。結(jié)果顯示，治療組卵囊數(shù)明顯少于對照組(p＜0.05)。治療的最佳劑量為1.5g/只鼠。
本發(fā)明的積極效果在于蛋白基因易于修飾和改造、穩(wěn)定性好、滲透性好、制備簡單、特異性強(qiáng)、殺滅作用強(qiáng)，對隱孢子蟲疾病具有很高的預(yù)防和治療作用。


圖1為重組質(zhì)粒的構(gòu)建策略圖。
具體實(shí)施例方式下列實(shí)施例旨在進(jìn)一步舉例描述本發(fā)明，而不是以任何方式限制本發(fā)明，在不背離本發(fā)明的精神和原則的前提下，對本發(fā)明所作的本領(lǐng)域普通技術(shù)人員容易實(shí)現(xiàn)的任何改動或改變都將落入本發(fā)明的待批權(quán)利要求范圍之內(nèi)。
實(shí)施例1從人工感染隱孢子蟲的犢牛糞便中用蔗糖密度梯度離心法分離純化微小隱孢子蟲卵囊(CSO)，經(jīng)超聲粉碎(100W×5min)后免疫BALB/C小鼠，應(yīng)用雜交瘤技術(shù)，制備出1株能穩(wěn)定分泌抗隱孢子蟲McAb的雜交瘤細(xì)胞株3D6，并通過免疫熒光法對其進(jìn)行了鑒定，其在子孢子表面呈彌慢性熒光反應(yīng)，提示其針對子孢子表膜。從分泌抗隱孢子蟲子孢子單克隆抗體的雜交瘤細(xì)胞3D6中，提取其mRNA，在反轉(zhuǎn)錄酶的作用下，合成cDNA，經(jīng)PCR擴(kuò)增(反應(yīng)條件94℃1min，55℃2min，72℃2min，30個循環(huán))獲得抗隱孢子蟲子孢子表膜單克隆抗體的輕、重鏈可變區(qū)基因VL和VH。在這兩個基因中加入連接肽基因后，經(jīng)進(jìn)一步PCR擴(kuò)增、組裝合成出單鏈抗體(ScFv)基因，將該基因克隆入pMD-18T載體后，進(jìn)行了序列測定。測序結(jié)果表明，獲得的單克隆抗體輕、重鏈可變區(qū)基因VL和VH符合小鼠抗體可變區(qū)基因特征，為功能性重排的鼠抗體可變區(qū)基因序列，分離得到的單鏈抗體基因ScFv由重鏈可變區(qū)基因VH-連接肽基因-輕鏈可變區(qū)VL基因構(gòu)成，這種構(gòu)成模式較VL-Linker-VH基因結(jié)構(gòu)具有更強(qiáng)的抗體親和活性。ScFv基因全長為720bp，其中VH基因?yàn)?51bp，編碼117個氨基酸，VL基因全長為324bp，編碼108個氨基酸。
將獲得的ScFv基因分別代替綠膿桿菌外毒素PE的受體結(jié)合區(qū)，利用原核表達(dá)載體pET28，構(gòu)建了用于表達(dá)重組毒素的原核表達(dá)質(zhì)粒3D6pET28-ScFv-PE(單鏈重組免疫毒素基因序列附后)。將上述質(zhì)粒轉(zhuǎn)化受體菌BL21(DE3)后，經(jīng)IPTG誘導(dǎo)，成功地表達(dá)了目的蛋白。重組毒素3D6ScFv-PE40的表達(dá)量占菌體蛋白總量的14％，表達(dá)產(chǎn)物以包涵體和上清兩種形式存在。將低溫誘導(dǎo)獲得的3D6ScFv-PE可溶性表達(dá)產(chǎn)物，加入含有脫囊后的子孢子的1640培養(yǎng)液中，觀察18h內(nèi)活動的子孢子數(shù)目和活動性的變化，結(jié)果與對照組相比，加入重組毒素組活動的子孢子數(shù)目明顯減少(P＜0.05)，活動性明顯降低，提示重組毒素對隱孢子蟲子孢子具有較強(qiáng)的殺滅作用，其治療隱孢子蟲病模型動物的劑量為1μg/只鼠。
實(shí)施例2從人工感染隱孢子蟲的犢牛糞便中用蔗糖密度梯度離心法分離純化微小隱孢子蟲卵囊(CSO)，經(jīng)超聲粉碎(110W×6min)后免疫BALB/C小鼠，應(yīng)用雜交瘤技術(shù)，制備出1株能穩(wěn)定分泌抗隱孢子蟲McAb的雜交瘤細(xì)胞株4G4，并通過免疫熒光法對其進(jìn)行了鑒定發(fā)發(fā)現(xiàn)針對子孢子表膜。從分泌抗隱孢子蟲子孢子單克隆抗體的雜交瘤細(xì)胞4G4中，提取mRNA，在反轉(zhuǎn)錄酶的作用下，合成cDNA，經(jīng)PCR擴(kuò)增(反應(yīng)條件95℃1min，56℃2min，72℃2min，30個循環(huán))，獲得抗隱孢子蟲子孢子表膜單克隆抗體的輕、重鏈可變區(qū)基因VL和VH。在這兩個基因中加入連接肽基因后，經(jīng)進(jìn)一步PCR擴(kuò)增、組裝合成出單鏈抗體(ScFv)基因，將該基因克隆入pMD-18T載體后，進(jìn)行了序列測定。測序結(jié)果表明，獲得的單克隆抗體輕、重鏈可變區(qū)基因VL和VH符合小鼠抗體可變區(qū)基因特征，為功能性重排的鼠抗體可變區(qū)基因序列，分離得到的單鏈抗體基因ScFv由重鏈可變區(qū)基因VH-連接肽基因-輕鏈可變區(qū)VL基因構(gòu)成。4G4 ScFv基因全長為717bp，其中VH基因?yàn)?48bp，編碼116個氨基酸，VL基因全長為324bp，編碼108個氨基酸。
將獲得的ScFv基因分別代替綠膿桿菌外毒素PE的受體結(jié)合區(qū)，利用原核表達(dá)載體pET28，構(gòu)建了用于表達(dá)重組毒素的原核表達(dá)質(zhì)粒4G4pET28-ScFv-PE(單鏈重組免疫毒素基因序列附后)。將上述質(zhì)粒轉(zhuǎn)化受體菌BL21后，經(jīng)IPTG誘導(dǎo)，成功地表達(dá)了目的蛋白。重組毒素4G4ScFv-PE40的表達(dá)量占菌體蛋白總量的12％，表達(dá)產(chǎn)物以包涵體和上清兩種形式存在。將低溫誘導(dǎo)獲得的4G4ScFv-PE可溶性表達(dá)產(chǎn)物，加入含有脫囊后的子孢子的1640培養(yǎng)液中，觀察20h內(nèi)活動的子孢子數(shù)目和活動性的變化，結(jié)果與對照組相比，加入重組毒素組活動的子孢子數(shù)目明顯減少(P＜0.05)，活動性明顯降低，提示重組毒素對隱孢子蟲子孢子具有較強(qiáng)的殺滅作用，其治療隱孢子蟲病模型動物的劑量為1.5μg/只鼠。
權(quán)利要求
1.抗隱孢子蟲重組免疫毒素蛋白的基因序列如下CAG GTG AAG CTG CAG GAG TCA GGA CCT GGC CTG GTG GCA CCC TCA 45Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser1 5 10 15CAG AGC CTG TCC ATC ACA TGC ACT GTC TCT GGG TTC TCA TTA TCC 90Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser16 20 25 30AGA TAT AGT GTA CAC TGG GTT CGC CAG CCT CCA GGA AAG GGT CTG 135Arg Tyr Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu31 35 40 45GAG TGG CTG GGA ATG ATA TGG GGT GGT GGA AGC ACA GAC TAT AAT 180Glu Trp Leu Gly Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn46 50 55 60TCA GCT CTC AAA TCC AGA CTG AGC ATC AGC AAG GAC AAC TCC AAG 225Ser Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys61 65 70 75AGC CAA GTT TTC TTA AAA ATG AAC AGT CTG CAA ACT GAT GAC ACA 270Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr76 80 85 90GCC ATG TAC TAC TGT GCC AGA AAC CGG TAT GGT AAT GCT ATG GAC 315Ala Met Tyr Tyr Cys Ala Arg Asn Arg Tyr Gly Asn Ala Met Asp91 95 100 105TAC TGG GGC CAA GGC ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC 360Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly106 110 115 120GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG GAC ATC GAG 405Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu121 125 130 135CTC ACT CAG TCT CCA GCC TCC CTA TCT GTA TCT GTG GGA GAA ACT 450Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly Glu Thr136 140 145 150GTC ACC ATC ACA TGT CGA GCA AGT GAG AAT ATT TAC AGT AAT TTA 495Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Tyr Ser Asn Leu151 155 160 165GCA TGG TAT CAG CAG AAA CAG GGA AAA TCT CCT CAG CTC CTG GTC 540Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val166 170 175 180TAT GCT GCA ACA AAC TTA GCA GAT GGT GTG CCA TCA AGG TTC AGT 585Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser181 185 190 195GGC AGT GGA TCA GGC ACA CAG TAT TCC CTC AAG ATC AAC AGC CTG 630Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu196 200 205 210CAG TCT GAA GAT TTT GGG AGT TAT TAC TGT CAA CAT TTT TGG GGT 675Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly211 215 220225ACT CCG TAC ACG TTC GGA GGG GGG AGC AAG CTG GAG CTG AAA CGG 720Thr Pro Tyr Thr Phe Gly Gly Gly Ser Lys Leu Glu Leu Lys Arg226 230 235 240CAT ATG GCC GAA GAG GGC GGC AGC CTG GCC GCG CTG ACC GCG CAC 765His Met Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His241 245 250 255CAG GCT TGC CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT CGC CAG 810Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln256 260 265 270CCG CGC GGC TGG GAA CAA CTG GAG CAG TGC GGC TAT CCG GTG CAG 855Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Thr Pro Val Gln271 275 280 285CGG CTG GTC GCC CTC TAC CTG GCG GCG CGG CTG TCG TGG AAC CAG 900Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln286 290 295 300GTC GAC CAG GTG ATC CGC AAC GCC CTG GCC AGC CCC GGC AGC GGC 945Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly301 305 310 315GGC GAC CTG GGC GAA GCG ATC CGC GAG CAG CCG GAG CAG GCC CTG 990Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Leu316 320 325 330GCC CTG ACC CTG GCC GCC GCC GAG AGC GAG CGC TTC GTC CGG CAG 1035Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln331 335 340 345GGC ACC GGC AAC GAC GAG GCC GGC GCG GCC AAC GCC GAC GTG GTG 1080Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val346 350 355 360AGC CTG ACC TGC CCG GTC GCC GCC GGT GAA TGC GCG GGC CCG GCG 1125Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala361 365 370 375GAC AGC GGC GAC GCC CTG CTG GAG CGC AAC TAT CCC ACT GGC GCG 1170Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala376 380 385 390GAG TTC CTC GGC GAC GGC GGC GAC GTC AGC TTC AGC ACC CGC GGC 1215Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly391 395 400 405ACG CAG AAC TGG ACG GTG GAG CGG CTG CTC CAG GCG CAC CGC CAA 1260Thr Glu Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln406 410 415 420CTG GAG GAG CGC GGC TAT GTG TTC GTC GGC TAC CAC GGC ACC TTC 1305Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe42l 425 430 435CTC GAA GCG GCG CAA AGC ATC GTC TTC GGC GGG GTG CGC GCG CGC 1350Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg436 440 445 450AGC CAG GAC CTC GAC GCG ATC TGG CGC GGT TTC TAT ATC GCC GGC 1395Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly451 455 460 465GAT CCG GCG CTG GCC TAC GGC TAC GCC CAG GAC CAG GAA CCC GAC 1440Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp466 470 475 480GCA CGC GGC CGG ATC CGC AAC GGT GCC CTG CTG CGG GTC TAT GTG 1485Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val481 485 490 495CCG CGC TCG AGC CTG CCG GGC TTC TAC CGC ACC AGC CTG ACC CTG 1530Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu496 500 505 510GCC GCG CCG GAG GCG GCG GGC GAG GTC GAA CGG CTG ATC GGC CAT 1575Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His511 515 520 525CCG CTG CCG CTG CGC CTG GAC GCC ATC ACC GGC CCC GAG GAG GAA 1620Pro leu Pro Leu Arg leu Asp Ala Ile Thr Gly Pro Glu Glu Glu526 530 535 540GGC GGG CGC CTG GAG ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC 1665Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg541 545 550 555ACC GTG GTG ATT CCC TCG GCG ATC CCC ACC GAC CCG CGC AAC GTC 1710Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val556 560 565 570GGC GGC GAC CTC GAC CCG TCC AGC ATC CCC GAC AAG GAA CAG GCG 1755Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala571 575 580 585ATC AGC GCC CTG CCG GAC TAC GCC AGC CAG CCC GGC AAA CCG CCG 1800Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro586 590 595 600CGC GAG GAC CTG AAG TAA CTG CCG CGA CCG GCC GGC TCC CTT CGC 1845Arg Glu Asp Leu Lys ***601 605AGG AGC CGG CCT TCT CGG GGC CTG GCC ATA CAT CAG GTT TTC CTG 1890ATG CCA GCC CAA TCG AAT ATG AAT TC
2.抗隱孢子蟲重組免疫毒素蛋白的基因序列如下CAG GTG AAG CTG CAG GAG TCT GGG GGA GGC TTA GTG AAG CCT GGA 45Gln Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly1 5 10 15GGG TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA TTC ACT TTC AGT 90Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Ala Phe Thr Phe Ser16 20 25 30AGC TAT GCC ATG TCT TGG GTT CGC CAG ACT CCG GAG AAG AGG CTG 135Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu31 35 40 45GAG TGG GTC GCA ACC ATT AGT GGT GGT GGT AGT TAC ACC TAC TAT 180Glu Trp Val Ala Thr Leu Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr46 50 55 60CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC AGA GAC AAT GCC 225Pro Asp Ser Val Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala61 65 70 75AAG AAC ACC CTG TAC CTG CAA ATG AGC AGT CTG AGG TCT GAG GAC 270Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp76 80 85 90ACG GCC ATG TAT TAC TGT GCA AGA CGG GAG GGC TGG TTT GCT TAC 305Thr Ala Met Tyr Tyr Cys Ala Arg Arg Glu Gly Trp Phe Ala Tyr91 95 100 105TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT 350Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly106 110 115 120TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG TAC ATC GAG CTC 395Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Ile Glu Leu121 125 130 135ACT CAG TCT CCA GCC TCC CTA TCT GCA TCT GTG GGA GAA ACT GTC 450Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr Val136 140 145 150ACC ATC ACA TGT CGA GCA AGT GGG AAT ATT CAC AAT TAT TTA GCA 495Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala151 155 160 165TGG TAT CAG CAG AAA CAG GGA AAA TCT CCT CAG CTC CTG GTC TAT 540Trp Tyr Gln Gln Lys Gly Gln Lys Ser Pro Gln Leu Leu Val Tyr166 170 175 180AAT GCA AAA ACC TTA GCA GAT GGT GTG CCA TCA AGG TTC AGT GGC 585Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly181 185 190 195AGT GGA TCA GGA ACA CAA TAT TCT CTC AAG ATC AAC AGC CTG CAG 630Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln196 200 205 210CCT GAA GAT TTT GGG AGT TAT TAC TGT CAA CAT TTT TGG AGT ACT 675Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Tyr211 215 220 225CCT CCC ACG TTC GGA GGG GGG ACC AAG CTG GAT CTG AAA CCG CAT 720Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Asp Leu Lys Pro His226 230 235 240ATG GCC GAA GAG GGC GGC AGC CTG GCC GCG CTG ACC GCG CAC CAG 765Met Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln241 245 250 255GCT TGC CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT CGC CAG CCG 810Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro256 260 265 270CGC GGC TGG GAA CAA CTG GAG CAG TGC GGC TAT CCG GTG CAG CGG 855Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Thr Pro Val Gln Arg271 275 280 285CTG GTC GCC CTC TAC CTG GCG GCG CGG CTG TCG TGG AAC CAG GTC 900Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val286 290 295 300GAC CAG GTG ATC CGC AAC GCC CTG GCC AGC CCC GGC AGC GGC GGC 945Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly301 305 310 315GAC CTG GGC GAA GCG ATC CGC GAG CAG CCG GAG CAG GCC CTG GCC 990Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Leu Ala316 320 325 330CTG ACC CTG GCC GCC GCC GAG AGC GAG CGC TTC GTC CGG CAG GGC 1035Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly331 335 340 345ACC GGC AAC GAC GAG GCC GGC GCG GCC AAC GCC GAC GTG GTG AGC 1080Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser346 350 355 360CTG ACC TGC CCG GTC GCC GCC GGT GAA TGC GCG GGC CCG GCG GAC 1125Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp361 365 370 375AGC GGC GAC GCC CTG CTG GAG CGC AAC TAT CCC ACT GGC GCG GAG 1170Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu376 380 385 390TTC CTC GGC GAC GGC GGC GAC GTC AGC TTC AGC ACC CGC GGC ACG 1215Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr391 395 400 405CAG AAC TGG ACG GTG GAG CGG CTG CTC CAG GCG CAC CGC CAA CTG 1260Glu Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu406 410 415 420GAG GAG CGC GGC TAT GTG TTC GTC GGC TAC CAC GGC ACC TTC CTC 1305Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu421 425 430 435GAA GCG GCG CAA AGC ATC GTC TTC GGC GGG GTG CGC GCG CGC AGC 1350Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser436 440 445 450CAG GAC CTC GAC GCG ATC TGG CGC GGT TTC TAT ATC GCC GGC GAT 1395Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp451 455 460 465CCG GCG CTG GCC TAC GGC TAC GCC CAG GAC CAG GAA CCC GAC GCA 1440Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala466 470 475 480CGC GGC CGG ATC CGC AAC GGT GCC CTG CTG CGG GTC TAT GTG CCG 1485Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro481 485 490 495CGC TCG AGC CTG CCG GGC TTC TAC CGC ACC AGC CTG ACC CTG GCC 1530Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala496 500 505 510GCG CCG GAG GCG GCG GGC GAG GTC GAA CGG CTG ATC GGC CAT CCG 1575Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro511 515 520 525CTG CCG CTG CGC CTG GAC GCC ATC ACC GGC CCC GAG GAG GAA GGC 1620leu Pro Leu Arg leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly526 530 535 540GGG CGC CTG GAG ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC ACC 1665Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr541 545 550 555GTG GTG ATT CCC TCG GCG ATC CCC ACC GAC CCG CGC AAC GTC GGC 1710Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly556 560 565 570GGC GAC CTC GAC CCG TCC AGC ATC CCC GAC AAG GAA CAG GCG ATC 1755Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile571 575 580 585AGC GCC CTG CCG GAC TAC GCC AGC CAG CCC GGC AAA CCG CCG CGC 1800Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg586 590 595 600GAG GAC CTG AAG TAA CTG CCG CGA CCG GCC GGC TCC CTT CGC AGG 1845Glu Asp Leu Lys ***601 605AGC CGG CCT TCT CGG GGC CTG GCC ATA CAT CAG GTT TTC CTG ATG 1890CCA GCC CAA TCG AAT ATG AAT TC
3.抗隱孢子蟲重組免疫毒素蛋白制備工藝包括以下步驟利用單克隆抗體技術(shù)和基因重組技術(shù)，從分泌抗微小隱孢子蟲子孢子表膜抗體的雜交瘤細(xì)胞株中克隆其可變區(qū)基因，再連接成單鏈抗體基因，然后將其插入原核表達(dá)載體的相應(yīng)位點(diǎn)上，構(gòu)建了重組原核表達(dá)質(zhì)粒，再分別采用酶切和PCR方法將去除受體結(jié)合區(qū)的細(xì)菌外毒素插入重組原核質(zhì)粒中單鏈抗體基因的下游位點(diǎn)，即利用單鏈抗體基因代替細(xì)菌外毒素的受體結(jié)合區(qū)，構(gòu)建重組表達(dá)質(zhì)粒；將上述構(gòu)建的重組表達(dá)質(zhì)粒分別轉(zhuǎn)化受體菌，經(jīng)IPTG誘導(dǎo)，SDS-PAGE分析表達(dá)產(chǎn)物，后對表達(dá)產(chǎn)物包涵體進(jìn)行純化和復(fù)性，并對其進(jìn)行體外抗蟲活性測定和模型動物隱孢子蟲病治療。
4.根據(jù)權(quán)利要求3所述的抗隱孢子蟲重組免疫毒素蛋白制備工藝，包括以下步驟從人工感染隱孢子蟲的犢牛糞便中用蔗糖密度梯度離心法分離純化微小隱孢子蟲卵囊，經(jīng)超聲粉碎后免疫BALB/C小鼠，應(yīng)用雜交瘤技術(shù)，制備出1株能穩(wěn)定分泌抗隱孢子蟲McAb的雜交瘤細(xì)胞株3D6，并通過免疫熒光法對其進(jìn)行了鑒定，其在子孢子表面呈彌慢性熒光反應(yīng)，提示其針對子孢子表膜，從分泌抗隱孢子蟲子孢子單克隆抗體的雜交瘤細(xì)胞3D6中，提取其mRNA，在反轉(zhuǎn)錄酶的作用下，合成cDNA，經(jīng)PCR擴(kuò)增，獲得抗隱孢子蟲子孢子表膜單克隆抗體的輕、重鏈可變區(qū)基因VL和VH，在這兩個基因中加入連接肽基因后，經(jīng)進(jìn)一步PCR擴(kuò)增、組裝合成出單鏈抗體ScFv基因，將該基因克隆入pMD-18T載體后，進(jìn)行了序列測定；將獲得的ScFv基因分別代替綠膿桿菌外毒素PE的受體結(jié)合區(qū)，利用原核表達(dá)載體pET28，構(gòu)建了用于表達(dá)重組毒素的原核表達(dá)質(zhì)粒3D6pET28-ScFv-PE，將上述質(zhì)粒轉(zhuǎn)化受體菌BL21后，經(jīng)IPTG誘導(dǎo)，成功地表達(dá)了目的蛋白。
5.根據(jù)權(quán)利要求3所述的抗隱孢子蟲重組免疫毒素蛋白制備工藝，包括以下步驟從人工感染隱孢子蟲的犢牛糞便中用蔗糖密度梯度離心法分離純化微小隱孢子蟲卵囊，經(jīng)超聲粉碎后免疫BALB/C小鼠，應(yīng)用雜交瘤技術(shù)，制備出1株能穩(wěn)定分泌抗隱孢子蟲McAb的雜交瘤細(xì)胞株4G4，并通過免疫熒光法對其進(jìn)行了鑒定發(fā)發(fā)現(xiàn)針對子孢子表膜。從分泌抗隱孢子蟲子孢子單克隆抗體的雜交瘤細(xì)胞4G4中，提取mRNA，在反轉(zhuǎn)錄酶的作用下，合成cDNA，經(jīng)PCR擴(kuò)增，獲得抗隱孢子蟲子孢子表膜單克隆抗體的輕、重鏈可變區(qū)基因VL和VH。在這兩個基因中加入連接肽基因后，經(jīng)進(jìn)一步PCR擴(kuò)增、組裝合成出單鏈抗體ScFv基因，將該基因克隆入pMD-18T載體后，進(jìn)行了序列測定；將獲得的ScFv基因分別代替綠膿桿菌外毒素PE的受體結(jié)合區(qū)，利用原核表達(dá)載體pET28，構(gòu)建了用于表達(dá)重組毒素的原核表達(dá)質(zhì)粒4G4pET28-ScFv-PE，將上述質(zhì)粒轉(zhuǎn)化受體菌BL21后，經(jīng)IPTG誘導(dǎo)，成功地表達(dá)了目的蛋白。
6.抗隱孢子蟲重組免疫毒素蛋白在預(yù)防和治療人和動物隱孢子蟲病的醫(yī)用用途。
全文摘要
本發(fā)明公開一種抗隱孢子蟲重組免疫毒素蛋白及其制備工藝和醫(yī)用用途，是一種預(yù)防和治療隱孢子蟲病的藥物，利用單克隆抗體技術(shù)和基因重組技術(shù)，從分泌抗微小隱孢子蟲子孢子表膜抗體的雜交瘤細(xì)胞株中克隆其可變區(qū)基因，再連接成單鏈抗體基因，然后將其插入原核表達(dá)載體的相應(yīng)位點(diǎn)上，構(gòu)建了重組原核表達(dá)質(zhì)粒，再分別采用酶切和PCR方法將去除受體結(jié)合區(qū)的細(xì)菌外毒素插入重組原核質(zhì)粒中單鏈抗體基因的下游位點(diǎn)，構(gòu)建重組表達(dá)質(zhì)粒。將上述構(gòu)建的重組表達(dá)質(zhì)粒分別轉(zhuǎn)化受體菌，經(jīng)IPTG誘導(dǎo)，SDS－PAGE分析表達(dá)產(chǎn)物，后對表達(dá)產(chǎn)物包涵體進(jìn)行純化和復(fù)性，并對其進(jìn)行體外抗蟲活性測定和模型動物隱孢子蟲病治療。
文檔編號A61P33/00GK1560066SQ20041001071
公開日2005年1月5日 申請日期2004年3月3日 優(yōu)先權(quán)日2004年3月3日
發(fā)明者張西臣, 李建華, 蔡輝 申請人:中國人民解放軍軍需大學(xué)