Cloning, Expression And Identification Of Escherichia Coli LTKA63, LTB Genes And Vibrio Cholerae CTB Gene

For lower immunization effects of genetic egineeing vaccines usually composed of a single protein antigen, it may be necessary to increase the immunogenicity of the vaccine antigens by co-administration of an appropriate adjuvant.Cholera toxin B subunit (CTB), one of the two kinds of peptides in enterotoxin produced by Vibrio cholerae, is generally accepted as a well-effected mucosal adjuvant. However, in the resently published data, recombinant CTB is little or failed to stimulate S-IgA responses towards admixed antigen, whereas strongly able to induce IgG in serum. The adjuvanticity of CTB required GMrbinding activity. Several previous studies demonstrated that CTB activates Th2 pathway, in which causing IL-4 production at a high level. It is well known that 1L-4 can selectly promote IgE synthesis and IgE imediates allergic reaction type I. Therefore, CTB was considered to be probably associated with IgE-imediated allergic reaction.E.coli heat-labile toxin (LT) is another very potent mucosal adjuvant but its application is limited because of the enterotoxicity. Molecular structure of LT, similar to CT, consists of one Asubunit (LTA) and five B subunits (LTB). LTA is an enzyme with ADP-ribosylating activity thatis responsible for the toxicity. LTB can bind the receptors GM] located on the surface of eukaryotic cell to mediate LTA entering target cells.LTK 63, a mutant by replacing serine with lysine at the 63th position in LTA peptide, was totally devoid of toxic effects in vitro and vivo, and it also acts as a strong mucosal adjuvant. Many recent studies showed that not only the LTA subunit, but also the LTB subunit alone has adjuvant activity. GMrbinding activity is also essential for the adjuvanticity properties of LTB. The adjuvanticity of LTA is unknown but exactly independent of ADP-ribosylation acticity. LT mainly stimulates Thl and secondarily Th2 responses, which induce IgG and mucosal IgA (S-IgA) but not IgE. It is interested that mix of LTA and LTB may enhane adjuvanticity because of their different mechanisms.Summarily, LTK63, LTKA63 and LTB are more suitable for the adjuvant in oral genetic engineering vaccine than CTB.MATERIALS AND METHODS1. Source of bacterial strain and cultureE.coli strain 44815 was inoculated on LB plate and incubated for 24 h at 37癈. A single colony on the plate was identified by Gram-staining method and microscopy. The bacterial strain was inoculated on LB plate again if the microbe was a Gram-negative, moderate-size bacillus. The inactivated culture by heating of Vibrio cholerae strain eastern 74 was offered by professor He Zhe-sheng2. Preparation of Bacterial Genomic DNAsThe routing Phenol-chloroform method was applied to prepare genomic DNAs of of E.coli strain 44815 and V.cholerae strain eastern 74. The DNA preparations was digested with DNase-free RNase and then extracted by Phenol-chloroform method. The DNAs used as templates in PCR were dissolved in TE buffer and the concentration and purification of the two DNA solutions were determined by ultraviolet spectrophotometry.3. PCROligonucleotide primers were designed to amplify the whole length of LTA, LTB and CTBgenes according to the published corresponding nucleotide sequences. The LTA and LTB genes from E.coli strain 44815 and CTB gene from V.cholerae strain eastern 74 were amplified by PCR, respectively. The results of PCR were observed after electrophoresis in 15 g L-1 agarose pre-stained with ethidium bromide.The sequences of primers for LTA gene amplification: 5'-CCG GAT ATC ATG AAA AAT ATA ACT TTC-3'(EcoRV, sense); 5'-CCG CTC GAG TAT TCA TAA TTC ATC CCG-3'(XhoI, antisense). The total volume per PCR was 100 l containing 2.5 mol L-1 each dNTP, 250 nmol' L-1 each the primers, 20 mol L-1 MgCl2, 3.0 U 3.0 U EX Taq polymerase, 100 ng DNA template and 1 PCR buffer (pH8.8). The parameters of PCR was described as the following: 94 5min, 1 cycle; 94 30sec, 50 30sec, 72 60sec, 10 cycles; 94 30sec, 50 30sec, , 72 70sec, 20 cycles (each of the following cycles a...