表达乙型脑炎病毒E蛋白重组减毒沙门菌活载体疫苗株的构建

目的:构建表达乙型脑炎病毒(JEV)E蛋白的口服重组减毒鼠伤寒沙门菌活载体疫苗株。方法:克隆JEV E基因,将其插入表达载体pYA3341中,构建重组质粒pYA3341-E,将重组质粒电转入鼠伤寒沙门菌疫苗株X4550(缺失asd、cya、crp基因),获得重组疫苗菌株X4550(pYA3341-E);鉴定重组菌E蛋白的表达,测定重组菌的稳定性、生长曲线、安全性,以及小鼠的免疫试验和血清中和试验。结果:酶切鉴定和序列测定证实重组质粒构建成功;SDS-PAGE检测有目的蛋白条带;Western印迹证实表达的E蛋白能与猪抗JEV阳性血清特异性结合;重组菌株在体外营养选择压力下,可稳定地携带重组质粒传代繁殖,在体内可较稳定地定居于肠系膜淋巴结和脾脏;小鼠口服试验证实重组菌无毒性作用,安全可靠;小鼠口服重组菌免疫,ELISA检测产生了抗JEV抗体;中和试验表明产生的抗体具有中和活性。结论:构建了能稳定表达JEV E蛋白的口服减毒鼠伤寒沙门菌疫苗株X4550(pYA3341-E),为研究乙型脑炎口服基因工程疫苗奠定基础。

Construction of the Recombinant Attenuated Salmonella typhimurium Live Vector Vaccine Strain Expressing JEV E Antigen and its Im- munogenicity Analysis

Objective：To construct a recombinant attenuated Salmonella typhimurium oral live vector strain expressing the E antigen of Japanese encephalitis virus（JEV）.Methods：The E gene of JEV was cloned and inserted into expression vector pYA3341.The recombinant plasmid pYA3341-E was electro-transformed into attenuated S.typhimurium vaccine strains X4550（Δcya,Δcrp,Δasd） to construct the live vaccine strains X4550（pYA3341-E）.The recombinant strain of X4500（pYA3341-E） was identified its E protein expression,stability,growth curve,safety in vitro and immunity evaluation by animal experiment in mouse.Results：With restriction enzyme digestion and gene sequencing,it was showed that the recombinant plasmid has been constructed successfully.SDS-PAGE and Western blot could detect the E protein band in S.typhimurium X4500（pYA3341-E） and the recombinant E protein could be recognized by the positive serum of JEV.The recombinant strain could propagate stably at noruishment selection pressure in vitro.The recombinant strain could colonize and persist stably in mesenteric lymph nodes and spleens of BALB/c mice.Its safety and reliability were verified by mouse experiment.The mice were immuned with recombinant strain by oral way,and the serum antibody could be detected by ELLISA.Conclusion：A recombinant oral live vaccine S.typhimurium strain X4500（pYA3341-E） which can express JEV E protein stably was constructed successfully.This live vaccine could be a basis to oral genetic engineering vaccine for JEV infection.