Cloning And Prokaryotic Expression Of CadF And Omp50 From Campylobacter Jejuni

Campylobacter jejuni (C.jejuni) is a major cause of gastrointestinal diseases worldwide. In addition, C.jejuni is also an important zoonosis pathogen.This pathogenic organism can cause many human clinical symptoms of campylobacteriosis including watery diarrhea, fever, abnormal cramps and hemorrhagic colitis.Furthermore, C.jejuni is also associated with Guillain-Barre syndrome and acute flaccid paralysis that may lead to respiratory muscle compromise and death.Eleven strains of Campylobacter jejuni,including six completed genomes and five draft genomes had been sequenced by the beginning of September,2009 in the world.In addition, ICDCCJ07001,whilch was also fininshed with completed genome, was sequenced by the Department of Communicable Disease Diagnostics(DCDD), National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. At the moment, people mianly focus on genomic information of single C.jejuni strain or genomic polymorphisms using gene microarray, while there was no analysis for single nucleotide polymorphisms (SNP) among multiple strains. Meanwhile, analsyis for SNP among multiple strains can furtherly understand the genetic polymorphisms of C.jejuni.Despite apparent importance of C.jejuni, there are no well antigen-antibody diagnose and commercial vaccine available for C.jejuni at present. Therefore it is necessary for us to exploit vaccine and well antigen-antibody diagnose technologies.Outer membrane proteins (OMPs), because they are surface exposed, are the most obvious targets for well antigen-antibody diagnose research and subunit vaccine research. Therefore our studies were to firstly analyse single nucleotide polymorphisms (SNP) of all single-copy protein coding sequences (CDSs) from eleven strains C.jejuni and then clone and express CDSs whilch their products are OMPs from C.jejuni.SNPs of CDSs form eleven C.jejuni strains were analysed through bioinformatics methods in this research. The bioinformatics methods included BLAST, CLUSTALW, Perl language programming and so on. Firstly multicopy CDS, whilch included intrastrain multicopy CDSs and multicopy CDSs among strains, were analysed. Then the non-redundant single-copy CDS set and the non-redundant multicopy CDS set were established. Finally all SNPs of single-copy CDSs were analysed and SNP database was established.The results showed there are 185 intrastrain multicopy CDSs and 99 multicopy CDSs among strains.There are 3729 CDSs in the non-redundant single-copy CDS set and 104 CDSs in the non-redundant multicopy CDS set. It was discovered that there are 589 strain-specifical CDSs and 94 CDSs with no SNP site. In addition, all other 3048 CDSs have 184707 SNP sites and the SNP percent is 7.15%.Aslo was discovered some other genetic information, including the SNPs for commen CDSs of eleven C.jejuni strains, SNPs for sup.doyleri delete CDSs, SNPs for 8 OMPs and so on.According to results of the single-copy CDS SNP analysis, Campylobacter adhesion to fibronectin (cadF) and outer membrane protein 50 (omp50) whose products are two important outer membrane proteins of campylobacter jejuni, were chose to clone and expressed in E. coli (DE3).At the beginning of the studies, signal peptides of both CadF and Omp50 were predicted by using SignalP 3.0 Server. Then primers were designed according to the terminals of the truncated cadF and omp50 respectively.Then amplified by PCR method and purified PCR products by TIANGEN midi purification kit.The purified PCR products were cloned into E. coli with pEASY-T1 Simple cloning vector. After identified by colony PCR and double restriction endonuclease digestion, the recombinant cloning vectors were extracted and digested by BamH I and Xho I.The purified target gene fragment were linked to pET30α(+) and the recombinant vectors were transformed into E. coli (DE3). Also identified by colony PCR and double restriction endonuclease digestion and additional sequencing analysis, the recombinant proteins rCadF and rOmp50 were induced and expressed in E. coli (DE3). The fusion proteins were identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF/TOF-MS. At last rabbit polyclonal antibodies were used to analyze the antigenicity of rCadF and rOmp50 by Western blot.This study successfully constructed a recombinant clone and expression vectors and recombinant proteins were successfully induced and expressed in E. coli (DE3). In addition SDS-PAGE and MALDI-TOF/TOF-MS demonstrated that the fusion proteins were correctly expressed.DNA sequencing for cadF and omp50 in recombinant expression plasmid pET30a(+)-cadF and pET30a(+)-omp50 respectively showed 100% homology with the formerly sequenced.rCadF was successfully purified under denaturing condition (8M urea) whereas rOmp50 wasn't. The Western blot trails demonstrated that both rCadF and rOmp50 are of antigenicity.In one word, lots of useful genetic information for C.jejuni are discovered from the work of the single-copy CDSs SNP analysis and provide experiment designment with clues and theoretical basises. The work of Cloning and Prokaryotic expression of cadF and omp50 was instrumental for future studies such as designment of rCadF-based or rOmp50-based diagnostic tools and vaccines.