Homologous Hsp20and Heterologous IL-10Expression In Protein Sec-secretion System Modified Bifidobacterium Longum NCC2705

Bifidobacteria, since founded in1906by Henry Tissier, has become a kind of important probiotics and its probiotic functional research has also been illuminated thoroughly. Bifidobacterum belongs to gram positive microbes that are generally recognized as safe (SAFE) due to its safety, they can survive during passing through the gastrointestinal tract and colonize in gastrointestinal tract. Bifidobacterium has become a kind of excellent oral live microorganism regulator in the gut. Taking advantage of the probiotic and biological characteristics, Bifidobacteria have the potential to deliver class of genetic engineering vaccine targeting at the gastrointestinal tract of host. To investigate the feasibility of using Bifidobacteria as oral vaccine vector, this study selected Bifidobacterium longum NCC2705strain as genetic engineering protein vaccine delivery vector or host, from several aspects, including the homologous gene expression, heterogonous gene expression, secretion system modification and its influence on secretion of heterogonous expressed protein, for the possible live delivery vaccine model genetic engineering probiotic strain construction, which were used to explore and evaluate the basic and practical application.Over-expression of homologous sHsp geneA small heat shock protein (sHsp) of Bifidobacterium longum, shp20gene family encoded proteins involved in cellular stress response, was cloned and constructed into an E. coli-Bifidobacteium shuttle vector pDP401with Bifidobacterial protein expressing cassette under control of Pgap Promoter and constrained by thup terminator, which was designated pDP401-sHsp. This expression vector was constructed to establish the system of electro-transformation and selection of B. longum NCC2705strain. pDP401-sHsp, the sHsp expression construction was successfully transformed into B. longum NCC2705, great influence on cellular physiological function elicited by over-expression of sHsp was evaluated in the transformant strain, and the function of sHsp in B. longum was first time investigated as well. In the sHsp homologous Over-expression recombinant strain, the cell growth rate, optical density and ability of acid production under condition of variety of abiotic stress (heat-stress, high temperature stress, acid and bile salt tolerance) were improved, compareing to the B. longum NCC2705wild strains. The result indicated that recombinant strain could survive from various abiotic stresses better than the wild strain. Up to now, the effective method to electrotransform protein expression cassette shuttle vector with antibiotic selection marker into B. longum, which was hard to transform vector into it for a long time, had been set up. Moreover, the targeted expression by above method has been proved to be functional.Expression of heterologous geneHuman IL-10gene ligated into pDP backbone based heterogonous protein-Bifidobacterial expression plasmid vector pDP870-IL10was constructed, without any codon modification and optimization, the IL-10was successfully expressed in the recombinant NCC2705strain, which was further confirmed by Western blot with IL-10specific antibody. The results suggested that, as a functional protein expressing host, to some extent, B. longum NCC2705is compatible and its tolerance to the different codon usage is varied within species. Based on above study, we proved that B. longum NCC2705can be used as an engineering strain to express heterologous gene, by which, target foreign genes could be deliver as the probiotic vaccine delivery host/agent.The protein secretion sec-system modification by heterolologous secDF expression in B. longum NCC2705SecD/F are components of many bacterial protein secretion sec-system, while most of lactic acid bacteria and Bifidobacteria are secDF natural deficiency organisms. Streptomyces coelicoler has a high ability to secret proteins, in which, the secDF encodes a fused secD-secF. The secDF of S. coelicoler was cloned constructed into the bifidobacterial protein expression plasmid PDP870-SecDF, and then transformed into B. longum NCC2705. Positive secDF NCC2705transformant was confirmed at DNA and expression levels. Through the sqRT-PCR, we observed that secDF expression in B. longum NCC2705transformant, and the secDF modified NCC2705strain was obtained.Effect of secretory expression of target protein by.sec-system modified B. longum NCC2705To evaluate the function of the target protein secreted by the sec-secretion system, we transformed the B. longum NCC2705strain by the constructed the plasmid co-expressing JL-10and secDF gene, the human IL-10gene and S. coelicoler-secDF co-expression vector pDP870-secDF-IL10. Western Blot indicated that the co-expression was successful. The expressing IL-10protein in the tranformant of strain with or without secDF and the result suggested that there is difference in between these two strains. Biological activity of IL-10 secreted by secDF-modified B. longum NCC2705strain was observed by inhibition of TNF-a induced inflammatory response of epithelial carcinoma HT-29cell line assay. Results indicated that B. longum secreted human IL-10could produce more than20%inflammatory inhibition rate, indicated by the IL-8expression level of HT-29cell. All of the results proved that the protein secretion system constructed in this study can directionally modify the target protein secretion state..Sec-secretion system modification is a method of practible and perspective usage for probiotic live vaccine delivery in intestinal tract circumstances.