传染性胰腺坏死病毒VP3蛋白的干酪乳杆菌表达系统构建

Construction of Expression System of Infectious Pancreas Necrosis Virus VP3 Protein in Lactobacillus casei

根据传染性胰腺坏死病毒(IPNV)VP3蛋白的全基因序列,设计并合成引物,以IPNV(ATCC VR-1318)细胞培养毒提取的核酸为模板,对传染性胰腺坏死病毒VP3蛋白的干酪乳杆菌表达系统进行了构建研究。结果显示:进行RT-PCR扩增得到截短的VP3基因约615 bp目的片段,将其克隆到pMD18-T Simple载体,经酶切、PCR扩增和序列测定后显示目的片段正确;将目的片段分别亚克隆到乳酸菌细胞表面表达型载体和分泌表达型载体,电转化于干酪乳杆菌,获得了阳性重组菌株。结果表明,通过本实验方法可构建表达传染性胰腺坏死病毒VP3蛋白的干酪乳杆菌表达系统,为实现IPNV VP3蛋白在乳酸菌中的表达及免疫原性研究奠定了基础。

Infectious pancreatic necrosis（IPN） virus, the truncated gene for the viral capsid protein（ VP3 ）, an approximately 615 bps fragment of IPNV gene VP3 that encompasses the major antigenic domains critical for neutralization, was cloned into the Lactobacillus casei vectors pPG1 and pPG2, and transformed into＇ L. casei 393 by eleetroporation, resulting in recombinant plasmids pPG1-VP3 and pPG2-VP3. The recombinant plasmids were detected by plasmid extraction, PCR, restriction enzymes digest and sequences analysis. The result indicated that a truncated form of VP3 of IPNV had inserted into the expression vectors and achieved Lactobacillus casei expression systems. It had elected the base for the expression of IPNV VP3 protein in L. casei and the production of oral vaccination.