摘要
【目的】中国控制猪瘟主要采用疫苗免疫,现有的猪瘟兔化弱毒疫苗有较好的免疫保护效果,但是不能从血清学上区分疫苗免疫猪与自然感染猪,猪瘟标记疫苗的研制与配套检测技术的开发可有效解决这一问题。【方法】利用猪瘟病毒低温诱变弱毒T株感染性克隆,将其全长结构蛋白基因替换为猪瘟病毒石门毒株全长结构蛋白基因,得到猪瘟石门重组病毒的全长感染性克隆pSMT;随后在pSMT E1蛋白的C端插入Flag抗原基因作为鉴别标记,构建猪瘟石门重组标记病毒的感染性克隆pSMT-Flag。经电转染法拯救出两株重组病毒vSMT和vSMT-Flag,通过SK6细胞进行病毒传代以获得重组病毒。【结果】多组酶切鉴定表明两株重组质粒成功构建,PCR扩增及测序、免疫过氧化酶单层细胞试验(IPMA)表明重组病毒已成功拯救且能成功传代。【结论】该标记病毒能够与Flag单抗发生特异性反应,且不影响E2蛋白中和表位与猪瘟单抗的结合,因此该毒株为研制CSFV新型标记疫苗提供了思路。
[ Objective ] Vaccination is still the main strategy for CSFV control in China. CSFV HCLV（Hog Cholera Lapinized Virus, HCLV） strain could protect effectively, but it couldn＇t be serologicaUy differentiated between infected pigs and vaccinated pigs （DIVA principle）, which could been effectively solved by marker vaccine and marker diagnostics. [ Method ] Based on infectious cDNA clone of CSFV T strain, which is a temperature-sensitive mutation strain, the complete structural protein-coding region of T strain had been replaced by that of ShiMen strain, and the recombinant plasmid SMT was constructed. Additionally, the Flag gene was inserted into C end of E1 protein of the recombinant virus as a positive marker, and the pSMT-Flag was constructed successfully. After electroporation, the recombinant viruses vSMT and vSMT-Flag were passaged by SK6 cell. [Result] Two recombinant plasmids were identified by specific restriction enzymes, the rescued viruses could be passaged successfully and confirmed by RT-PCR, sequencing and immunoperoxidase monolayer assay（IPMA）. [Conclusion] This marker virus could react with the Flag specific monoclonal antibody while could not influence the combination between E2 protein and its specific monoclonal antibody, which could be a potential CSF novel marker vaccine.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第1期187-194,共8页
Scientia Agricultura Sinica
基金
国家自然科学基金(30571377)
关键词
猪瘟
重组病毒
FLAG
标记疫苗
classical swine fever
recombinant virus
Flag
marker vaccine
