Construction And Immunogenicity Assay Of Co-expression Recombinant Plasmids Including EtpA And LT B From Enterotoxigenic Escherichia Coli

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen of diarrhealdisease in humans and animals in developing countries. ETEC has negative effects onlive-stock farming, fish breeding, aviculture, and human health. The development of anETEC vaccine has been hindered by the heterogeneity of known molecular targets and lackof broad-based sustained protection from existing vaccine strategies. However, the newlyidentified ETEC two-partner secretion locus directs the secretion of high-molecular-weightglycosylated protein EtpA, along with the putative EtpB transporter, stimulates theadhesion of ETEC to epithelial cells. LT B may have a complex function in ETECpathogenesis because it facilitates adhesion to intestinal epithelial cells in vitro andpromotes small intestinal colonization in vivo.In this paper, we report the cloning of the EtpA and LT B genes from ETEC usingpolymerase chain reaction (PCR). The target genes were inserted into the plasmid vectorpEASY-Blunt. The recombinant plasmids were confirmed using PCR, enzyme analysis,and sequencing. This recombinant plasmids pEB-EtpA, pEB-LT B and pET-32a weredigested with BamHI and XhoI. The fragments were then identified and cloned into theprokaryotic expression vector pET-32a. This recombinant plasmids pET-EtpA and pET-LTB were transformed into E. coli BL21(DE3) and induced to expression with IPTG. Theproducts of expression were analyzed by SDS-PAGE. We obtained the optimum expressionconditions of pET-EtpA and pET-LT B. The maximal expression quantity of EtpA proteinwas observed at5h,37°C, and1mM IPTG, whereas that of LT B protein was observed at6h,37°C, and1mM IPTG. We also were used to construct recombinant eukaryoticexpression plasmid pcDNA-EtpA and pcDNA-LT B. We have been identified the accuracyof recombinant eukaryotic expression plasmid pcDNA-EtpA and pcDNA-LT B byPT-PCR.An indirect ELISA for detecting antibodies to EtpA protein or EtpA/LT B proteins orpcDNA-EtpA plasmids or pcDNA-EtpA/pcDNA-LT B plasmids was established by usingpurified adhesion protein for coating antigens. The conditions of reaction were as follows:the optimal coating antigen concentration of EtpA protein antigens was1:160. The optimaldilution of corresponding serum samples was1:200. It was proved that the methed waswell specific and repeatable by cross experiment, blocking test.The humoral and cellularimmune responses induced by the recombinant plasmids pcDNA-EtpA and pcDNA-LT B were analyzed in mice. The results showed that after two intramuscular injections, specificantibodies to EtpA present in serum of mice. The splenocytes proliferation of miceimmunized with recombinant plasmids, stimulated by concanavalin A (ConA) orrecombinant EtpA, were distinctly increased. The results of challenge protect assays andthe maternal antibodies in mice showed that the pcDNA-EtpA DNA vaccine and thepcDNA-EtpA/pcDNA-LT B DNA vaccine which possessed excellent immunogenicitycould provide an protection against ETEC. This study provides a foundation for the deeplydevelopment of ETEC vaccine.