Research Of Expression And Immunological Character Of V-Recombinant Toxins Gene For Food Poisoning Bacteria

Food poisoning is viewed as a significant public health concern.Especially bacterial food poisoning is one of forms often happened and hasbeen an increasing focus of public health in recent years. It is important toprotect food from risk of contamination and to prevent food poisoning. ThePoisons produced by some of these bacteria such as Vero toxin, SEs andBoNTs are important pathogen of causing serious foodborne illness. Theyhave a very small infections dose and so they are vital to detect andeliminate these pathogen from foods. These toxins may be produced in thefoodstuff or within the intestine, to produce symptoms very soon afteringestion. With the development of of Molecule Biology and MoleculeGenetics, the current knowledge on these pathogen and the ongoingresearch of the structure of toxin genes, pathogenic and virulence traits,methods of detection and their application are more and more beingdeepened. Nowadays, in the study of detection of food poisoning bacteria,one is to search quick and exact technology ,the other is to seekbroad-spectrum detection methods. Because general assay is not only takelong time but also cannot meet the quest of detecting a large quantity offoods. There is no extensive and quick methods about detection of foodpoisoning bacteria. There are only two broad-spectrum but not mature assaymethods including protein chips and DNA chips. In this study, the structure gene sequence of VT1 and VT2B, SEA andSEB, BoNTs were respectively amplified from E.coli O157:H7 andStaphylococcus aureus and C.botulinum by PCR with the primers designedaccording to their published gene sequences or Genebank. After beingcloned into pUCm-T vector, the amiplified genes was sequenced andanalyzed. The sequenced results are consistent with the published genesequences or Genebank. RHDS-5 fusion gene was constructed by PCR via a linker sequenceencoding five amino acids (G-P-G-P-G) and connecting cloned five toxinsgene frag-ments. After digested with restriction nuclease NcoI and XhoI, thefusion gene was inserted into expression vector pET22b(+). The resultantrecombinant plasmid, terming RHDS-5-22b, was transformed into E.coliDH5α. The amiplified v-combinant toxins genes was sequenced andanalyzed. The sequenced results are consistent with the published genesequences or Genebank. Sequence encoding the mature fusion protein oftoxin gene of RHDS-5 are 2937bp, encoding 979 amino acids. The weightof recombinant fusion protein is 112.369 kDa. The resultant recombinantplasmid, may be used to express. The amplified v-combinant toxins fusion gene was linked intoprokaryotic expression vector pET22b. Then the recombinant RHDS-5-pET22b was transformed into host strain E.coli BL21 (DE3) induced byIPTG. The expressed protein of (112.369kDa) was identified by SDS-PAGE.With the time extending, the amount of the expressed protein increasedafter induced by IPTG(1mmol/L) by thin-layer scan and amounted to9.90% of the total protein of E.coli BL21(DE3)after induced byIPTG(1mmol/L) for 4 hours. The pattern of expression was soluble(5.295%)and inclusion body (4.694 %)at 37℃after induced byIPTG(1mmol/L) for 4 hours, and the expressed protein was all soluble at25℃after induced by IPTG(1mmol/L) for 10 hours. Most of solubleexpressed proteins were in cytoplast and small parts of them in cellperiplasm. The RHDS-5 inclusion body protein was denatured and renatured.Thesoluble expressed protein was purified by SDS-PAGE and proteinextracting liquicds.They immunized respectively"JiRong"rabbits to prepareanti-serum. The prepare anti-serum reacted with five toxins test. The resultof ELISA showed that obvious positiv reaction with VT1B,VT2B,SEA,SEB and Hc and their antibody value are Hc 800×,VT151200×,SEA 800×,VT2 25600×,SEB 400×respectively.The different deposition lineappeared in agar diffusion test.These results showed that the rabbitantibodies raised against the recombinant fusion protein could recognize thenatural toxin. The results indicated that recombinant toxins kept theantigenicity of natural toxins in different antibody potent. In addition, theresults of antibody specificity ELISA assays did not demonstrated obviouspositive reaction with 27 bacteria or toxins such as Salmonella enteritidis,Listeria and other serum type of toxins. The result of ELISA Sensitivityindicated that theirs sensitivity degree are Hc 31.25ng/mL,VT1 3.75ng/mL,SEA 125.00ng/mL,VT2 7.50ng/mL,SEB 62.50ng/mL.It indicated thatRHDS-5 antigen has special antigenicity. They indicated that the two typesexpressed proteins all effectively induced the body to produce the antibodyand had better antigenicity. This experiment revealed that a few of toxinsprotein gene via a linker sequence may be expressed in prokaryotic...