The Predicition Of The Promoter Functional Sequence Of Lactobacillus Enolase And Its Functions Identification

Lactic acid bacteria, which is generally recognized as safe microorganism and important flora in the animal intestines, and it plays an important role in maintaining the balance of intestinal microbial flora, improving constipation, preventing diarrhea and improving the immunity of the body. These probiotic characteristics make it a key host candidate strains for exogenous antigen protein expression, delivery and intestinal immunity. At present, the expression vector of lactic acid bacteria is mainly inducible expression vector, which expression of exogenous protein production is low and not conducive to the production practice, thus limiting its application. Modify the cis-acting element which regulate gene expression is an effective way of improving protein expression, while the promoter occupies a key position in regulation of bacterial transcription level, so the constitutive promoter which can express the target protein stably and sustainably is a very important expression element for construct the expression vector. In this study, Lactobacillus casei is the research object, by Lactobacillus casei genome c DNA microarray analysis, and find the constitutive promoter which applicable in expression of heterologous protein.The Lactobacillus casei was continuously cultured in MRS medium with glucose, lactose, galactose, trehalose, fructose, sucrose, fructo-oligose and raffinose different carbohydrate as the carbon source, and 5 times were transferred. Total RNA were extracted, after the reverse transcription, microarray analysis the whole genome c DNA, with 16 S RNA as housekeeping gene. The result showed that enolase gene is the highly expressed gene in eight kinds of sugar as carbon source of Lactobacillus casei. Based on this, we speculate that Lactobacillus casei enolase gene contains a constitutive promoter. Through the analysis of the upstream gene sequence of enolase gene and the bioinformatics software, two putative promoter sequences were identified, which were named P776 and P342 respectively. The recombinant expression plasmids p PG-P342 EGFP and p PG-P776 EGFP were obtained by connecting P776 and EGFP with reporter gene P342, and then connected with the expression plasmid pPG-T7g10-PPαT which has removed promoter. In order to verify the predicted promoters strength, the HCE promoter of the expression plasmid pPG-T7g10-PPαT were compared, the EGFP gene was connected to expression plasmid pPG-T7g10-PPαT and constructed the recombinant expression plasmid pPG-T7g10-EGFP.The recombinant expression plasmid p PG-P342 EGFP, p PG-P776 EGFP and p PG-T7g10-EGFP were transformed into Lactobacillus(L)casei 393 by electroporation respectively, and construct the recombinant strains p PG-P776EGFP/L. casei 393, p PG-P342EGFP/L. casei 393 and pPG-T7g10-EGFP/L. casei 393. The expression of report gene EGFP was detected by the analysis of Western blot, fluorescence quantitative PCR, flow cytometry and confocal laser experiments.Construction of recombinant Lactobacillus expression results showed that the three recombinant lactic acid bacteria strains which were composed of different promoters were observed green fluorescence by confocal laser microscope, the Western blot test report gene encoding EGFP expressed correctly, about the size of a 26 kDa, which indicate d that the prediction P342 and P776 promoter both has promoter activity. In order to compare P342, P776 and HCE promoter of original expression vector of the strength. RNA was extracted when the recombinant strains in logarithmic growth phase, and then reverse transcription to c DNA as a template, using chloramphenicol(Cm) gene as housekeeping gene for relative quantitative PCR. Results showed that efficiency ratio of the three promoters HCE:P342:P776 is 1:0.8:1.8; According to western blot relative quantitative analysis, the expression level of the p PG-P776EGFP/L.casei 393 is the highest of the three recombinant strains; Flow cytometry analysis showed that the fluorescence signals intensity of L. casei 393, pPG-P342EGFP/L. casei 393, pPG-P776EGFP/L. casei 393 and pPG-T7g10-EGFP/L.casei393 was 30.2%, 38.5%, 52.2% and 45.2% respectively; pPG-P342EGFP/L. casei 393 and pPG-P776EGFP/L. casei 393 cultured 12 h, 18 h, 24 h, 30 h and 36 h, Western blot analysis indicated that the P342 and P776 promoter canactivated protein expression sustainably, and expression of maximum amount was 24h; p PG-P342EGFP/L. casei 393 and p PG-P776EGFP/L. casei 393 cultured in MRS media with glucose, fructose, galactose, lactose, sucrose, trehalose, raffinose, fructo-oligose as carbon source repectively. Western blot results showed that constitutive promoter P776 and P342 can drive the expression of EGFP in different sugars as carbon source in the culture medium, and promoter P776 was stronger than HCE promoter, this study lay the foundation for construction efficient lactic acid bacteria expression vector.