表达鼠精子Cyritestin蛋白的减毒沙门氏菌疫苗株的构建及其鉴定

目的 构建表达鼠精子eyritestin蛋白的可口服减毒沙让氏菌活疫苗株。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yA3149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X4632和X4550中，以获得重组菌株X4632（pCFL)和X4550（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。X4550(pCFL)在体内可较稳定地害居于Peyer‘s结，肠系膜淋巴结和脾脏；X4632（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 X4632（pCFL),X4550（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。

Construction and identification of the attenuated Salmonella typhimurium strains expressing mouse sperm cyritestin

Aim To construct a live oral Salmonella typhimurium vaccine strain expressing mouse sperm cyritestin. Methods The gene encoding the mature cyritestin protein was obtained by digestion of the plasmid TAZ83/13 using restrictive enzyme and was inserted into the plasmid pYA3149. The resulting recombinant plasmid pCFL was transformed into the delta cya, delta crp, delta asd attenuated Salmonella typhimurium vaccine strains X4632 and X4550, to construct the live vaccine strains X4632(pCFL) and X4550(pCFL). Results The recombinant plasmid pCFL was stable in the X4632(pCFL) and X4550(pCFL)strains under culture condition. The recombinant strain X4550(pCFL)could colonize and persist stably in Peyer's patches, mesenteric lymph nodes and spleens of BALB/c mice. The X4550(pCFL) could also colonize and persist stably in Peyer's patches. Both strains could express recombinant mature cyritestin protein recognized by the anti cyritestin monoclonal antibody and had no toxicity. Conclusions The live vaccine strains X4632(pCFL)and X4550(pCFL)are worthy to be studied as a contraceptive vaccine.