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目的 构建表达鼠精子cyritestin蛋白的可口服减毒沙门氏菌活疫苗株。方法 应用质粒TAZ83/ 13,将之进行双酶切获得含有成熟cyritestin蛋白cDNA序列的酶切片段。将其插入表达载体 pYA314 9中 ,构建重组质粒pCFL。将该重组质粒转入鼠沙门氏伤寒杆菌疫苗株X4 6 32和X4 5 5 0中 ,以获得重组菌株X4 6 32 (pCFL)和X4 5 5 0 (pCFL)。结果 该两种无抗药性的重组菌株在体外营养选择压力下 ,可较稳定地携带重组质粒传代繁殖。X4 5 5 0 (pCFL)在体内可较稳定地定居于Peyer’s结、肠系膜淋巴结和脾脏 ;X4 6 32(pCFL)可较稳定地定居于Peyer’s结。二者均可表达被抗cyritestin单抗 (mAb)识别的重组cyritestin蛋白。口服该疫苗菌株无明显的毒性作用。结论 X4 6 32 (pCFL)、X4 5 5 0(pCFL)疫苗株的构建 ,为研究以cyritestin为靶抗原的精子抗原避孕疫苗打下了基础
Objective To construct an orally inactivated attenuated Salmonella live vaccine strain expressing mouse sperm cyritestin protein. Methods Plasmid TAZ83 / 13 was used to double-digest the cDNA of mature cyritestin protein. This was inserted into expression vector pYA3149 to construct recombinant plasmid pCFL. The recombinant plasmids were transformed into Salmonella typhimurium vaccine strains X4 6 32 and X4 5 0 0 to obtain recombinant strains X4 6 32 (pCFL) and X4 5 5 0 (pCFL). Results The two non-drug-resistant recombinant strains can be more stable to carry the recombinant plasmid in vitro propagation under the pressure of selective nutrition. X4 5 50 (pCFL) colonizes Peyer’s knot, mesenteric lymph node and spleen in vivo more stably; X4 6 32 (pCFL) colonizes Peyer’s knot more stably. Both can express recombinant cyritestin protein recognized by anti-cyritestin monoclonal antibody (mAb). Oral administration of the vaccine strain showed no significant toxic effects. Conclusion The construction of XCF6 vaccine (pCFL) and X4 5 5 0 (pCFL) vaccine lays the foundation for the study of sperm antigen contraceptive vaccine with cyritestin as target antigen