摘要
为了构建表达猪瘟病毒E2蛋白的口服重组减毒鼠伤寒沙门氏菌活载体疫苗株,亚克隆猪瘟病毒E2基因,将其插入到表达载体pYA3341中,构建了重组质粒pYA3341-E2。将该重组质粒电转入鼠伤寒沙门氏菌疫苗株X4550(缺失asd、cya、crp基因),获得重组疫苗菌株X4550(pYA3341-E2),并对重组菌表达的E2蛋白进行SDS-PAGE和Western-blot分析、测定其在体内外的稳定性、生长曲线、安全性及动物免疫试验。结果显示,重组菌能表达E2蛋白且表达的蛋白能与猪瘟病毒阳性血清特异性结合。在体外营养选择压力下,重组菌株在体外可稳定地携带重组质粒传代繁殖,在体内可稳定地定居于肠系膜淋巴结和脾。小鼠口服试验证实重组菌无毒性,安全可靠。动物免疫试验表明,口服重组菌免疫猪产生了抗E2蛋白抗体。淋巴细胞增殖试验表明,重组菌能诱导机体产生较强的细胞免疫应答。结果表明,成功构建了能稳定表达猪瘟病毒E2蛋白的口服减毒鼠伤寒沙门氏菌疫苗株,为研究猪瘟口服基因工程疫苗奠定了基础。
In order to construct a recombinant attenuated Salmonella typhimurium oral live vector strain expressing E2 antigen of classical swine fever virus（CSFV）,the CSFV E2 gene was subcloned and inserted into the expression vector pYA3341.The recombinant plasmid pYA3341-E2 was finally electro-transformed into delta cya,delta crp,delta asd attenuated S.typhimurium vaccine strain X4550 to construct the live vaccine strain X4550（pYA3341-E2）.The recombinant X4550（pYA3341-E2） strain was identified from E2 protein expression,stability,growth curve,safety in vitro and immunity evaluation by animal experiment in swine.SDS-PAGE and Western-blotting analyses showed that the E2 protein expressed in the S.typhimurium X4550（pYA3341-E2） could be recognized by the positive serum of anti-CSFV.The recombinant strain could stably propagate at noruishment selection pressure in vitro.The recombinant strain X4550（pYA3341-E2） could colonize and persist stably in mesenteric lymph nodes and spleens of BALB/c mice with its safety and reliability verified by mouse experiment.The recombinant strain X4550（pYA3341-E2） was immunized orally to swine and the E2 protein serum antibody could be detected.Lymphocyte proliferative response results indicated that the recombinant strain can also induced an obvious cellular immune response in swine.Results showed that a recombinant oral live vaccine S.typhimurium strain X4550（pYA3341-E2） which can express CSFV E2 protein stably was constructed successfully,and this live vaccine could be a basis to oral genetic engineering vaccine of CSF.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2011年第9期901-906,共6页
Chinese Veterinary Science
基金
国家大学生创新性实验计划项目(2010)
西北农林科技大学青年学术骨干项目(E111020901)
陕西省农业科技创新项目(2010NKC-06)
关键词
猪瘟病毒
E2蛋白
减毒鼠伤寒沙门氏菌
疫苗
classical swine fever virus
E2 protein
attenuated Salmonella typhimurium
vaccine
