Construction In A Mutant Strain Of Capmylobacter Jejuni With Deleting NeuB1 Gene And Evaluation For Its Clinical Importance

PART ICONSTRUCTION AND IDENTIFICATION IN A MUTANTSTRAIN OF CAPMYLOBACTER JEJUNI WITHDELETING NEUB1 GENE[Objective] On the basis of constructing and identifying a mutant of Campylobacter jejuni(CJ) O:19 strain with deleting neuBl gene and absence of sialic acid(N-acetylneuraminic acid, NANA) in outer core of lipoogliosaccharide (LPS) by molecular cloning technology and homologous recombination, to demonstrate the important role of NANA in the pathogenesis of CJ-associated Guillain-Barre syndrome(GBS).[Methods] (1) Construction and identification of the mutant vector: DNA fragment neuBl::KmR consisting of KmR cassette and the flanking neuB 1 intragenic sequence was amplified by PCR from the genomic DNAof an neuBl mutant enteritis-associated CJ O:2 strain. The purified PCR product was cloned into the suicide vector pGEM-T to construct the mutant vector pGEM-neuBl::KmR which was identified by endonuclease digestion and PCR; (2) Construction and identification of the mutant strain: by natural transformation the mutant vector was transformed into a wild O:19 CJ strain which was selected from four CJ strains. PCR, RT-PCR and DNA sequencing of PCR product were respectively used for confirming the mutant strain. After 25 consecutive propagation of the mutant strain, PCR and RT-PCR were conducted for testing its stability; (3) LPS was analyzed by SDS-PAGE and the NANA of LPS was determined by acidic ninhydrin reaction and periodate-resorcinol reaction; (4) Cholera toxin B subunit (CTB) binding assay was utilized for detecting GMl-like epitope in the whole-cell lysates of the strains.[Results] (1) PCR product of 2259bp was harvested. The DNA fragment neuBl::KmR was successfully cloned into the pGEM-T vector, which was identified by endonuclease digestions and PCR; (2) Construction and identification of mutant strain with deleting neuBl gene: A deleting neuBl mutant from a GBS-associated CJ O:19 strain was selected out with kanamycin. The lengths of PCR product of the mutant and its corresponding wild strain were about 2130bp and 630bp respectively, and the difference in length between the two products wasequal to that of the KmR cassette. By DNA sequencing, it was further confirmed that the KmR cassette was inserted into the neuB 1 gene of CJ O:19 strain already and no influence on the neuBl downstream genes by the exerted insertion. Loss of the transcriptional activity of neuBl gene in the mutant strain was confirmed by RT-PCR. After 25 consecutive propagation, the mutant strain still kept the resistance to kanamycin and the absent transcriptional activity of neuBl gene; (3) LPS molecular weight of the mutant strain was smaller than that of the wild strain in SDS-PAGE analysis. Structure of sialic acid was well demonstrated by both acidic ninhydrin reaction and periodate-resorcinol reaction in the LPS of wild strain but not in the mutant LPS; (4) Lysate and LPS of the wild strain could be binded to CTB but not for those of the mutant strain.[Conclusion] (1) By the suicide vector pGEM-T and molecular cloning technology, neuBl mutant vector pGEM-neuBl::KmR has been successfully constructed from DNA of a neuBl mutant enteritis-associated CJ O:2 strain; (2) The neuBl mutant of GBS-associated CJ O:19 strain has been successfully constructed by method of homologous recombination; (3) NANA of LPS of the mutant strain was taken out; (4) In contrast with the wild LPS, ganglioside GMl-like epitope, NANA, was absent. It was indicated that molecular mimicry between CJ LPS and GM1 had been destructed due to the absence of NANA structure; (5) The supposition thatmolecular mimicry and cross-reaction were involved in the pathogenesis of CJ-induced GBS was demonstrated from the view of molecular structure due to the successful construction of the CJ mutant deleting neuBl gene. NANA in LPS outer core ogliosaccharide and the epitopes of gangliosides such as GM1 on peripheral nerves both could be the key antigens to activate the cross-immune process; (6) The mutant strain could be a better candidate for fur...