Cloning,Expression And Preparation Monoclonal Antibody Against FLA,PEB1 Of Campylobacter Jejuni

Campylobacter jejuni have emerge over the last three decades as significant clinical pathogens and is currently one of the leading causes of bacterial diarrhoea in the world, therefore presents a significant challenge to public health.Human infection with C.jejuni is associated with postinfectious complications including arthritis,Reiter syndrome and Guillain-Barre syndrome.They are also found in a wide range of sites in animals with some causing infections of the reproductive tract of certain domestic species which can lead to either abortion and infertility.To optimize cultural conditions of C.jejuni,the growth condition was investigated under the different medium,temperature,growth time and preservation,respectively.The results showed the optimal cultural conditions are as follows:5%O₂,10%CO₂,85%N₂,37-42℃,culture time 36-48h.Using this optmid culture condition,the row chicken,frozen chiken,milk and waste water samples were collected for isolating the C.jejuni.The result showed the average C.jejuni isolation rate was 23.75%for raw chicken,11.25%for frozen chicken,16.50%for cow milk and 6.25%for waste water in nanjing market.Traditional methods for the detection of C.jejuni is time-consuming and false negative results are likely to occur.Conventional PCR and nested PCR detection were developed based on the flaA and peblA genes of C.jejuni.While the Traditional methods can detect C. jejuni with a limit at 45-100CFU/mL,conventional PCR can be used to detect C.jejuni at as low as 10-16 CFU/mL and nested PCR sensitivity reaches 2-6 CFU/mL.The results show the sensitivity of nested PCR are superior to that of traditional detection methods and Conventional PCR.Based on theflaA and peblA genes of C.jejuni in GenBank,two pairs of primer were designed and the flaA and peblA genes were amplified by PCR from C.jejuni (ATCC29428) genomic DNA.The PCR fragments were ligated into the pMD18-T vector and sequenced.The sequences were compared to the flaA and peblA genes of different kinds of C.jejuni strains announced at GeneBank to analyze their homology and diversity degree.The result showed that flaA and peblA genes are about 88.3%and 98.1%identical to the reported C.jejuni NCTC11168.In order to construct eukaryotic expression vectors pET-28a(+)-flaAg and pET-28a(+)-peblAg to obtain bioactive FLA and PEB1,we optimized and synthesized the DNA fragment of flaAg and peblAg gene of C.jejuni in accordance with the E.coli's codon preference,and inserted them to the expression vector pET-28a(+).The recombinant expression plasmids pET-28a(+)-flaAg and pET-28a(+)-peblAg were transformed into E.coli BL21(DE3),incubated at 37℃,4h later,OD600 reached 0.6,the transformants were induced for expression of recombinant protein by IPTG..After denaturation, purification by Ni²⁺ affinity chromatography and renaturation.,the expreesion proteins both of the recombinant FLA and PEB1 proteins were over 70%of total bacterial protein in form of inclusion bodies,and SDS-PAGE analysis indicated that molecular weight of recombinant proteins were about 25 kDaa and 29kDaa.Westernblot indicated that both of the recombinant FLA and PEB1 could react with polyclonal antibody of C.jejuni. Bioactive protein.FLA and PEB1 were successfully expressed in E.coli BL21(DE3).To prepare monoclonal antibodies(McAbs) against C.jejuni,Babl/c mice were immunized with the inactivated whole C.jejuni ATCC29428 cell.After fusing and cloning,the hybridoma cell lines secreting monoclonal antibodies against C.jejuni FLA protein were screened by regular cell fusion and subcloning approach.The specificities of these monoclonal antibodies were determined by ELISA and Western blotting.Three hybridoma cell lines secreting monoclonal antibodies(McAbs) against C.jejuni FLA protein were produced and designated as 1G8,1G9 and 3F2.