表达猪细小病毒VP2蛋白的重组干酪乳杆菌的构建

利用Oligo设计合成一对引物P1、P2,分别含有BamHI和XhoI酶切位点,以猪细小病毒株LJL12的DNA为模板,采用PCR技术扩增VP2基因,克隆到pMD18-TSimple载体,并进行酶切、PCR鉴定和序列测定。将VP2基因分别亚克隆到干酪乳杆菌细胞表面表达型载体pPG1和分泌型表达载体pPG2的BamHI和XhoI酶切位点,电转化干酪乳杆菌Lactobacillus casei393,获得阳性重组菌株。成功构建了猪细小病毒VP2蛋白的干酪乳杆菌表达系统,命名为pPG1-VP2/L.casei393和pPG2-VP2/L.casei393。

Construction of Lactobacillus casei Expressing Recombinant Classical Swine Fever Virus T Cell Epitope and Porcine Parvovirus VP2 Protein

Lactobacillus casei 393 was selected as a bacterial carrier for the development of live mucosal vaccine to express porcine parvovirus(PPV) VP2 protein.A pair of primers were designed containing BamHI and XhoI sites by Oligo,respectively.With PPV strain LJL12 genome as template,the VP2 gene was amplified by PCR and cloned to pMD18-T simple plasmid,and the recombinant plasmid pMD18-T-VP2 was identified by enzyme digested,PCR and sequencing.Then the VP2 gene was sub-cloned to BamHI and XhoI sites of cell surface expression plasmid pPG1 and secretion expression plasmid pPG2,named pPG1-VP2 and pPG2-VP2.The recombinant plasmid was electrotransformed into Lactobacillus casei 393 generating pPG1-VP2/L.casei 393 and pPG2-VP2/L.casei 393.