Clone Of CheY,ciaB,dnaJ,cfrA Genes From Campylobacter Jejuni And Construction Of Their Eukaryotic Expression Vector

Campylobacter jejuni (C. jejuni) as the human food born pathogenic bacteria,is one of the major causes of acute infection of gastrointestinal tract. Besides,it is closely related to human acute inflammatory demyelinating polyneuropathy,that is Guillain-Barre syndrome,which is the significant cause of acute flaccid paralysis. Recent years,as the booming of molecular biotechnology and genetic engineering, human begin to study the mechanisms of molecular biology more and more. Especially,as the full genome sequence of C.jejuni NCTC11168 (serotype penner O:2) published in 2000,the study of C.jejuni pathogenic mechanism in molecular level has a new perspective. C.jejuni mainly causes disease by the mechanism of invading, adhering, colonization, toxin producing and molecular simulation. Researches have shown that the pathogenicity of C. jejuni is the combined action of many virulence factors.Objective: In this reseach, the cluster of C. jejuni penner O:19 lulei strain isolates from north China has been proved can cause GBS.14 genes related to invading,adhering,colonization or toxin producing have been chosen to TA clone and test sequence.Then the tested sequences were compared to the corresponding genes of C.jejuni NCTC11168 to observe their similarity indices. 4 genes of higher conservatism and immunogenicity among these genes heve been chosen to clone ,and insert into the eukaryotic expression vector .Then get the eukaryotic expressions,and make a foundation for the genetically engineering vaccine of C.jejuni.Methods: amplification of target genes: The template was the cluster of C. jejuni penner O:19 lulei strain which has been proved can cause GBS.14 target genes named grpE, dnaK, chew, cheY, cheV, flaA, cdtA, cdtB, cdtC, peb2, pebC, ciaB, dnaJ and cfrA were amplificated by polymerase chain reaction(PCR).TA clone: 14 target genes were linked to pGEM-T Easy vector singly to complete TA clone,and then used the competence of Escherichia coli(Top10 or DH5a)to complete inversion.After cultivating the bacteria, plasmids were gained. The plasmids were detected by gel electrophoresis which have been cut by the restriction enzyme. The plasmids were also detected by testing sequence.Then the tested sequences were compared to the corresponding genes of C.jejuni penner O:19 lulei strain and NCTC11168 using DNASTAR software to observe their similarity indices.Construction the eukaryotic expression vector:4 genes nemed cheY, ciaB, dnaJ and cfrA which have higher conservatism and immunogenicity heve been chosen to amplificated by pyrobest polymerase chain reaction.These 4 genes were used to construct the eukaryotic expression vector using pEF neo-myc vector and which heve the coincident restriction enzyme cutting site as KpnⅠand EcoRⅤor KpnⅠand SpeⅠ.After the inversion of the competence of Escherichia coli(Top10 or DH5a),the plasmids were gained and the sequence were tested.The 293T cells were used to transfection and Western Blotting were used to test the express of the eukaryotic expression vector.Results :The target genes of grpE, dnaK, chew, cheY, cheV, flaA, cdtA, cdtB, cdtC, peb2, pebC, ciaB, dnaJ were amplificated and TA cloned successfully. The target gene of cfrA was amplificated and TA cloned by two pieces because of its long sequence.After cultivating the bacteria, gaining plasmids and testing sequence, the tested sequences were compared to the corresponding genes of C.jejuni penner O:19 lulei strain and NCTC11168 using DNASTAR software to observe their similarity indices.The results like these: the similarity indices of dnaK, chew, cheY, cheV, cdtA, cdtB, cdtC, peb2,pebC, ciaB, dnaJ, cfrA-1 genes are among 95.1% and 100%, the similarity index of flaA is 92.6%, the similarity index of grpE is 89.9%,and the similarity index of cfrA-2 is below 50%. Using pEF neo-myc vector, genes of cheY, ciaB, dnaJ and cfrA were constructed the eukaryotic expression vector and expressed successfully. The expressions of CheY′, CiaB′2 and DanJ′Proteins were detected by Western Blotting. But the expressions of CfrA-1′and CfrA-2′Proteins were not detected.Conclusions:14 target genes related to the mechanism of invading, adhering, colonization and toxin producing of C. jejuni were amplificated and TA cloned successfully. The tested sequences were compared to the corresponding genes of C.jejuni penner O:19 lulei strain and NCTC11168 using DNASTAR software to observe their similarity indices.The majority similarity indices are high which explained their higher conservatism. 4 genes with higher conservatism and immunogenicity were onstruct the corresponding eukaryotic expression vector,which will make a foundation for the genetically engineering vaccine of C.jejuni in the future.