抗隐孢子虫重组免疫毒素蛋白及其制备工艺和医用用途

摘要

本发明公开一种抗隐孢子虫重组免疫毒素蛋白及其制备工艺和医用用途，是一种预防和治疗隐孢子虫病的药物，利用单克隆抗体技术和基因重组技术，从分泌抗微小隐孢子虫子孢子表膜抗体的杂交瘤细胞株中克隆其可变区基因，再连接成单链抗体基因，然后将其插入原核表达载体的相应位点上，构建了重组原核表达质粒，再分别采用酶切和PCR方法将去除受体结合区的细菌外毒素插入重组原核质粒中单链抗体基因的下游位点，构建重组表达质粒。将上述构建的重组表达质粒分别转化受体菌，经IPTG诱导，SDS－PAGE分析表达产物，后对表达产物包涵体进行纯化和复性，并对其进行体外抗虫活性测定和模型动物隐孢子虫病治疗。

主权项

1、抗隐孢子虫重组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 Ser 1 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 45 GAG 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 215 220 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 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