Construction Of Recombinant Lactobacillus Casei Expression LTAK63and LTB Of E.coli And Mucosal Adjuvanticity Evaluation

Enterotoxigenic Escherichia coli (ETEC) are an important pathogens causing infant andmammal diarrhea. The heat-labile enterotoxin (LT) secreted by ETEC is the major pathogenicfactor of diarrhea for infant animals. The LT protein is composed of two subunits coded by anoperon; toxic subunit A (LTA) is a28kDa enzyme and non-toxic subunit B (LTB) is a60kDaprotein, composed of five identical polypeptides (11.6kDa), contain the cellular receptor bindingfunction. The LT, LTA and LTB are a potent signalling molecule capable of modulating cellularimmunity response and humoral immunity response, and the mechanism of LTA and LTB asadjuvant are different, suggesting they can work together. Despite their immunomodulatory effect,wild-type LT and LTA are toxic and unsuitable for clinical.Based on the published nucleotide sequence of LTA and LTB, primers were designed andsynthesized. First, the gene of LTA mature peptide was PCR amplified from the chromosomal DNAof ETEC cvcc230strain, the LTAK63t and LTAK63genes were joined by using overlap extensionPCR (SOE-PCR) technique. The LTB and rsLTB genes were amplified by PCR using the plasmidpPG-2-LTB, and the rsLTB fragment contains RBS sequence and signal peptide sequence (ssUSP).All of the interest genes were cloned into plasmid pMD18-T simple followed enzyme digestion andsequence analysis, then getting the recombinant plasmids. Second, We construct recombinantpET-28a-LTA/BL21(DE3)pLysS, LTA was produced in BL21(DE3)pLysS and purified. The micewere immunized three times by rLTA, serum of mice was collected after the immunization, and theantiserum of mouse had higher ELISA titer (1:12800). While,6weeks BALB/c mice wereimmunized three times with purified rLTB protein. Two hybridoma clones secreting antibodyagainst rLTB were obtained by fusing the splenocytes of immunized mice with SP2/0myelomacells. With purified rLTB as detecting antigen, McAbs against rLTB were prepared and positivehybridoma clones were screened by indirect-ELISA. The two McAbs belonged to IgG1. The resultsof Western Blot showed two McAbs can specifically recognize pentamer LTB protein, andconfirmed the McAbs had practical value.The fragments of LTAK63and LTB were cloned into Lactobacillus expression vector pPG-2,and the positive was named pPG-2-LTAK63-LTB. The fragments of LTAK63t and rsLTB werecloned into lactobacillus expression vector pPG-2, and the positive was named pPG-2-LTAK63t-rsLTB. The recombinant plasmids pPG-2-LTAK63-LTB and pPG-2-LTAK63t-rsLTB were electrotransformed into Lactobacillus.casei393(L.casei393), and then obtained the expressionrecombinant pPG-2-LTAK63-LTB/L.casei393and pPG-2-LTAK63t-rsLTB/L.casei393. Therecombinant strains were induced by1%lactose. Because the expression amounts were too low, thetwo recombinant strains were not conspicuously expressed by SDS-PAGE. The results of WesternBlot indicated that the expressed protein possessed the antigenic specificity which could berecognized by mouse anti-LTA serum and mouse anti-LTB serum, the expression product ofrecombinant pPG-2-LTAK63-LTB/L.casei393was about38KDa, the expression product ofrecombinant pPG-2-LTAK63t-rsLTB/L.casei393were about27KDa and11KDa.To identify the value of the recombinant L.casei393as mucosal adjuvant on immune response,40(SPF) BALB/c mice were assigned to4groups. The first group was orally immunized byrecombinant pPG-2-LTAK63-LTB/L.casei393, the second group was orally immunized byrecombinant pPG-2-LTAK63t-rsLTB/L.casei393, and the third group was orally immunized byrecombinant pPG-2/L.casei393, while the three groups were orally immunized by recombinantpPG-2-FaeG/L.casei393. The forth group was only orally immunized by recombinant pPG-2/L.casei393, as a blank control. Serums, vaginal wash, nasal wash of mice were collected beforeimmunization and on the7th,14th,21thday after the immunization. Fecal pellets were collectedbefore immunization and Odd-numbered days after the immunization, until27thday. The antibody,sIgA and IgG, specific for FaeG were determined. Immunized mice’ lymphocyte proliferation testwere explored by MTT in vitro on the28thafter the immunization. Protection experiment ofimmunized mice was also explored. All the results showed that the group given recombinantpPG-2-LTAK63-LTB/L.casei393and pPG-2-LTAK63t-rsLTB/L.casei393were higher than notgiven, the two recombinant strains L.casei393could improve cellular immunity response andhumoral immunity response, and the added recombinant pPG-2-LTAK63t-rsLTB/L.casei393groupwas more higher than added recombinant pPG-2-LTAK63-LTB/L.casei393group.The study confirmed that the recombinant strains (This study) used with the model antigenrecombinant strain could enhance mucosal immune and system immune response, indicated therecombinant strains pPG-2-LTAK63-LTB/L.casei393and pPG-2-LTAK63t-rsLTB/L.casei393could exhibit mucosal Adjuvanticity. All theses work established a good foundation for the study ofmucosal adjuvant.