猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白在干酪乳杆菌中的共表达及免疫小鼠特异性抗体的测定

Co-expression of CSFV T cell epitope E290 peptide and PPV VP2 protein in Lactobacillus casei and determination of specific antibodies in immunized mice

摘要:

将分别编码猪瘟病毒T细胞表位E290多肽和猪细小病毒主要保护性抗原VP2蛋白的重组基因插入干酪乳杆菌分泌型表达载体pPG中,构建了重组表达载体pPG-VP2-E290,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白的乳酸菌共表达系统,经2%乳糖在MRS培养基中的诱导表达,对诱导表达的菌体及培养上清液进行SDS-PAGE检测表明,有约70kDa蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot分析结果表明所表达的蛋白具有与天然病毒蛋白一样的抗原特异性。以诱导表达上清液作为抗原进行的间接ELISA实验也表明,重组的目的蛋白获得了分泌表达。将该重组干酪乳杆菌经口服接种途径免疫BALB/c小鼠,收集粪便样品检测小鼠产生抗PPV的特异性sIgA抗体,采集血液样本检测血清中抗PPV及抗E290的特异性IgG。结果表明分泌型的重组菌pPG-VP2-E290/L.casei 393免疫小鼠能够产生明显的抗体水平,为重组猪瘟与猪细小病毒乳酸菌口服活菌疫苗的研制奠定了重要的物质基础。

Abstract:

Lactobacillus casei strain 393 was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant classical swine fever virus (CSFV) T cell epitope E290 peptide and porcine parvovirus (PPV) VP2 protein. The recombinant genes encoding CSFV T cell epitope E290 peptide and PPV VP2 protein, respectively, were cloned into the secretion expression vector pPG, and then the pPG-VP2-E290 was electrotransformed into L.casei 393 giving rise to recombinant strain pPG-VP2-E290/L.casei 393. The recombinant L.casei 393 was induced by 2% lactose in MRS and about 70kDa protein was detected with SDS-PAGE in induced recombinant strain and culture supernatants. The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The indirect ELISA test also indicated that the interest protein was expressed and secreted from the recombinant strain. Specific anti-PPV VP2 secret immunoglobulin A (sIgA) antibody was detected by indirect ELISA in the feces, anti-PPV VP2 and anti-CSFV E290 peptide immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice after intragastric administration. The results indicated that the mice immunized with recombinant strain pPG-VP2-E290/L.casei393 could produce clear antibody level, which establish important material basement for the development of lactic acid bacteria oral vaccine of recombinant CSFV and PPV.