Construction Of Constitutive Lactobacillus Expression Vector And Comparison Of The Expression Effect

pPG612is a kind of Lactobacillus/E.coli shuttle expression vector, while its expression level was low, target genes can be expressed only under the existence of inducers, it’s complicated to apply in the production, according to this, this study was based on the optimized pPG612vector to construct a constitutive surface display expression vector with high expression level of foreign protein without adding inducers.Clostridium perfringens is one of the major pathogens which can cause a variety of animals’ necrotizing enterocolitis, intestinal toxemia, human and animal traumatic gas gangrene and food poisoning of human, pathogenic factor is the exotoxin it produces. All types of Clostridium perfringens can produce a toxin, which is one of the most important pathogenic toxins of Clostridium perfringens, the researches at home and abroad about which have been deepened to seek better treatment, for this, this experiment selected a detoxicated protein as the detection protein of the reconstructed vector.In this experiment, we built a lactobacillus casei constitutive expression vector with a toxin protein expressed on the surface of recombinant bacteria without inducers. On the basis of pPG612vector, the original elements were replaced to HCE promoter, PgsA anchor, rrnBT1T2terminator, the reconstructed vector was named pPG-PPT, the a toxin gene fragment was inserted into the expression vector pPG-PPT and obtained the recombinant expression vector pPG-PPaT, and was transformed into L.casei393by electroporation, and got the a toxin protein expressed on the surface of the recombinant Lactobacillus casei. By the analysis of Western blot, indirect ELISA, indirect immunofluorescence and confocal laser experiments, it showed that a toxin protein was expressed on the surface of the recombinant bacteria. In order to optimize the constitutive expression vector to increase the amount of protein expression level and the stability of mRNA in the recombinant bacteria, we try to use two kinds of enhancers which have been applied to prokaryotic and eukaryotic protein expression, translational enhancer T7g10enhancer and Ω enhancer, these two enhancers were synthesized by Sangon (Shanghai) according to the relevant references, inserted into the downstream of HCE promoter, constructed two new recombinant Lactobacillus casei vectors pPG-Ω-PPαT and pPG-T7g10-PPαT expression system, and through Western blot, indirect ELISA, fluorescence quantitative PCR, flow cytometry, indirect immunofluorescence and confocal laser experiment analysis, and compare its a toxin protein expression level with the one expressed in the original vector pPG-PPaT. According to Western blot, pPG-T7g10-PPα17L.casei393has the highest expression level of a toxin protein. By the analysis of indirect ELISA, the difference between the contrast bacteria and the three recombinant bacteria is significant (P<0.01), the expression level in the pPG-Ω-PP α TIL.casei393is about2folds compared to pPG-PP α T/L.casei393, the expression level in the pPG-T7g10-PP α T/L.casei393is about11folds compared to pPG-PP a T/L.casei393. According to the mRNA level of qPCR, the difference between the contrast bacteria pPG-PPα TIL.casei393and recombinant bacteria pPG-Ω-PP α TIL.casei393, pPG-T7g10-PP α T/L.casei393is significant (P<0.01), by the analysis of REST2009, the expression level in the pPG-Ω-PP α T/L.casei393is about2folds compared to pPG-PP α T/L.casei393, the expression level in the pPG-T7g10-PP α T/L.casei393is about9folds compared to pPG-PP α T/L.casei393. According to the result of flow cytometry, the percentage of the bacteria with fluorescence signals in the total bacteia, pPG-PPT/L. casei393is0.7%, pPG-PP α T/L.casei393is8.5%, pPG-Ω-PP α T/L.casei393is10.9%, pPG-T7g10-PP α T/L.casei393is15.8%, increasingly one by one, the a toxin protein expression level expressed on the surface of recombinant bacteria is also increasing one by one, pPG-T7g10-PPaT/L.casei393has the highest expression level of a toxin protein, the difference between the contrast bacteria and the three recombinant bacteria is significant (P<0.01). It shows that the yellow and green fluorescence appeared on the surface of bacteria pPG-T7g10-PPα T/L.casei393is most. Both of1FAT and CLSM results showed that a toxin protein expressed on the surface of recombinant bacteria.In this study, we also prepared the polyclonal antibody against PgsA anchor for the identification of the PgsA anchor-α toxin fusion protein by Western blot. The antibody titer is1:51200.Through the comparison analysis of the three expression vectors, a toxin protein expression level of the recombinant lactobacillus casei pPG-T7g10-PPαT/L. casei393was the highest and has the best expression effect.These results indicate that although a toxin protein has got expressed in the recombinant lactobacillus casei expression system pPG-PPαT/L.casei393, its expression level was not ideal, the expression yields were low and less stable, while the a toxin protein expression levels have got a significant improved in the inserted translation enhancer T7g10and Ω enhancer expression vector pPG-Ω-PPαT and pPG-T7g10-PPαT, the expression of a toxin protein has been significantly improved, and the stability of recombinant plasmid expression vector in L.casei393inserted enhancer T7g10and protein expression status was relatively stable, but the stability of plasmid with Ω enhancer was not so stability as the vector with T7g10. As a conclusion, restructuring lactobacillus casei pPG-T7g10-PPT/L,.casei393protein expression system can provide a reference for creating the constitutive surface expression system expressing exogenous proteins without inducers, and the recombinant lactobacillus casei can obtain high expression level of heterologous proteins by fermentation tank culture, compared with the inducible expression system, it has simple operation and can reduce industrial cost-production, its application prospects will be broader.