Recombinant Cloning And Expression Of The A,D Gene S Protein Of TGEV And Escherichia Coli. Heat-labile Enterotoxin B Subunit Gene

Transmissible gastroenteritis of swine (TGE) is an acute contagious disease which can lead to vomiting, dewatering and serious diarrhea. It caused by transmissible gastro-enteritis virus belonging to Coronaviridae Coronavirus genus(TGEV). Enterotoxigenic E.coli (ETEC) is a important entero-pathogenic bacteria which can cause young pig diarrhea, young pig yellow dysentery and young pig white flux. According to the publication of GenBank TGEV protein S, A, D gene sequence and E.coli heat-labile enterotoxin B subunit (LTB) gene sequence, we design a pair of primer, using RT-PCR technology to amplification S₁ gene containing A, D site and LTB gene, insert each of them to pET-28a(+)to construction prokaryotic expression plasmid, uesing PCR, restriction identification and sequence determination. After being induction and expressing the recombined bacterium, we uesing SDS-PAGE and Western-blot to detect the expression product.Fragment of S₁ gene were amplified by reverse transcription-polymerase chain raction (RT-PCR). This genes total length is 980 bp, coding 262 amino acids. Sequence analysis shows that the autoploidy between S₁ gene and the gene sequence of publication in GenBank reach to 97 percent, the autoploidy between amino acids sequence is more than 95 percent, it confirm different strain of S₁ gene has high conservatism. The expression products is about 33.7 ku and most are inclusion;Western-blot analysis to make clear that the S₁ protein has reactionogenicity, can be identified by TGEV masccline serum. Similarly, LTB gene were amplified by polymerase chain raction (PCR), the total length is 375 bp, coding 124 amino acids, Sequence analysis shows that gene the autoploidy between LTB gene and the gene sequence of publication in GenBank reach to 99 percent, the autoploidy between amino acids sequence is more than 98 percent. The expression protein of LTB is about 14 ku.Western-blot analysis suggests LTB protein also has reactionogenicity.Redesign a primer, make the nonsense codon of LTB gene TAG mutate to TAC, to add to a EcoRI restriction site at its downstream. Redesign another primer, introduction a EcoRI restriction site into the upstream of S₁ gene, using the amplification products of S₁ gene and LTB gene as template, amplified by polymerase chain raction (PCR) again, restriction and connecting to recomposition into LTB₂-S_1-2 gene. The total length of gene is 1371 bp, coding 461 amino acids. Inserted LTB₂-S_1-2 into pET-28a(+), to construction recombine prokaryotic expression plasmid. Induction and express to obtain fusion protein is about 45 ku and mainly as inclusion body. Western-blot analysis shows that the expressed interest protein can be identified by TGEV and LTB masccline serum, the expression product is specific protein. This experiment provides the material basis for the further research about recombine LTB₂-S_1-2 gene fusion protein at the field of mucous membrane immunization, it will develops the production of safe and efficient engineering combined vaccine for TGEV and ETEC gene.