Research Of Expression And Immunological Character Of Poly-Recombinant Flagellae Gene For Vibrio Parahaemolyticus

As the improvement of food quality and the prosperity of the industry, our society focus more attention on the food safety, food poisoning is the common problem that we meet in food safety. However, the most common food poisoning is the bacterial-poisoning. Nowdays, vibrio-poisoning often take place in littoral aera of summer and fall which vibrio parahaemolyticus as represent. People pay attention to the vibrio-poisoning because of high frequency and great harm. How to avoid vibrio-poisoning is a urgent topic. At present, through unique gene to accredit bacteria have became the best method in medical diagnosis and food inspection. It is quicker﹑faster and more sensitive than ordinary method. The poly-recombined flagellae may be an innovation which will be broad-spectrum and quick in bacteria-poisoning.In this experiment we are used genome DNA of Vp to template, base gene order come from publication and genenbank to design primer, FlaA﹑FlaB and FlaF gene of Vp are amplificated according to synthetic primer. After purification to connect to pMD-18T vector and transform into E.coli DH5α. Through masculine clone bolting, those are sequenced to deliver company after PCR and enzyme cutting identification. The result of sequencing is 100% homology compare with gene order come from publication. Through overlap extension PCR method, the clone gene fragments were ligated in series with the linker sequences to obtain a single band peptide chain of FlaA—FlaF—FlaB, the head and tail of sequncing use NdeI and XhoI restricted enzyme site, the ending codon is TAA. Tandem peptide chain ligate with pET-22b(+) after enzyme cutting and transform into E.coli DH5αto get monoclone, then transform into DE3, masculine clone are identificated to sequencing throngh NdeI and XhoI enzyme cutting identification. The result of sequencing is 99% homology. The mutated site in sequencing are 753,1575,2199,2682. Sequence are GTA→GTC, TCA→TCG, ATT→ATC,GGT→GGC in order, but theencoding frame was right. The length of the gene F is 3420bp, encoding 1140 amino acid residues. The molecular weight is 119ku through DNAsis software analysis. The recombinant plasmid F could express successful. The gene engineering bacteria pET-22b-F are fermented on bulk with use IPTG to induce to express and optimize express condition. Through SDS-PAGE electrophoretic analysis, the result display: expression quantity is the highest in 37℃and IPTG induce to express 4h.In interest protein position appear visible expression, the size is correspond with prediction molecular weight(119ku) as well as expression quantity is the highest in 1mmol/L IPTG. Thin-layer scanning display the mass of expression foreign protien accout to 23.5% in the mass of all tropina, the most express protien is inclusion body protein,the little is dissolubility protien.The inclusion body protein of expressed poly-recombinant flagellae F are degenerated treatment then through SDS-PAGE electrophoresis and electroeluting method for purification, the dissolubility protien are condensed, then through SDS-PAGE electrophoresis and utilize protein leaching liquor to purify, and determine the content of protein. We immunized the JiRong rabbit every 7 days and the total time was 35 days which use the common method. After immunizing, we collected the blood from the heart and produced the anti-serum.The acquired anti-serum undertake immunological reaction with soluble antigen. The result is evident positive reaction through ELISA detection which explain the prokaryotic expressed protien F could stimulate organism to produce antibody efficiently and possess immunogenicity favourable. The acquired anti-serum carry out immunological reaction with 27 no-target bacteriums. The result is not evident positive reaction through ELISA detection. But have evident reaction with target bacterium which explain the poly-recombinant flagellar antibody have genus specificity favourable.The sensitivity of ELISA methodis: Vp 0.7×105/mL, VA 1.26×105 /mL, VMet2.08×105/mL, VF1.05×105/mL, VMin0.74×105/mL, VV0.97×105/mL.The result of Vp and VMin are better than others. The experimental result explain: prokaryotic expression vector could series connection three protein gene at least. The overall length could arrive 3400bp.The recombination protein gene could contain two linker but not effect expression of every protein. So the antibody of the poly-recombinant flagellar have had meaning of broad-spectrum antibody.The sensitivity test and agar diffusion test establish theory and practice foundation that there can be used in the food poisoning bacteria and construct the board-spectrum,quick,special detecting ways with poly-recombinant flagellae.