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Research on the Fuction of the fljA-Like Gene of Salmonella Enterica Serovar Typhi

Author: ZouZuo
Tutor: HuangXinXiang
School: Jiangsu University
Course: Clinical Laboratory Science
Keywords: Salmonella typhi fljA fliC z66 antigen j Antigen
CLC: R378.23
Type: Master's thesis
Year: 2009
Downloads: 47
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Abstract


Objective: It has long been that the Salmonella typhi single-phase strains that only the phase Ⅰ the flagellin coding genes fliC z66 positive Salmonella typhi found two flagellin encoding gene, z66 antigen-coding genes (fljB: z66) and fliC, after the z66 antigen encoding gene, there is a gene, phase Ⅱ flagellin encoding gene cluster in fljA similar, the purpose of this article is by study the fljA like gene of Salmonella typhi bacteria whip Mao Suji due fliC expression regulation understanding its function. Methods: (1) secreted proteins and bacterial protein analysis using trichloroacetic acid - acetone extraction the Salmonella typhi wild-type strain (GIFU10007), fljA like gene deficient strain (Wt △ fljA) secreted proteins and antibody-induced strain (007j ~) SDS-PAGE and identification, and Western blot analysis of secreted proteins and bacterial protein; gene expression (2) fljA kind fljA like genes both ends of the sequence design cited material z66 antigen-positive typhoid Salmonella bacteria gene group as a template, by PCR obtain the gene, and expression of the recombinant transformed to E. coli TG1, the connection of the vector pBAD / g Ⅲ induced culture, recombinant plasmid into the Salmonella typhi j ~ strain with the gene expression of the L-arabinose induction fljA sample; (3) fliC gene transcription level expression analysis by real-time quantitative RT-PCR observation fljA-like gene on fliC transcription level regulation; (4) Preparation of fliC :: lacZ mutants using suicide plasmid-mediated homologous recombination prepared Salmonella typhi beta - Galactosidase insertion mutant fliC :: lacZ design Kpn Ⅰ and Sal Ⅰ digestion site-specific primers fliC gene was amplified by PCR on the downstream homologous DNA fragment and with Kpn Ⅰ and Sal Ⅰ the fragments digested pSV-β-galactosidasevector (promoterless lacZ fragment) directional connection, and cloned into the suicide vector pGMB151 the Sma I, then by electroporation into Salmonella typhi j ~ strains, in sucrose plates containing X-Gal obtained by screening homologous recombination mutant fliC :: the lacZ, this strain is a translational fusion strains; (5) fliC gene translation level expression analysis to observation fljA sample by measuring the activity of the β-galactosidase of LacZ gene expression of FliC translation level adjustment. Results: (1) by SDS-PAGE and Western blot identification and analysis of secreted proteins and bacterial protein found the the wild strains GIFU10007 expression FljB: z66 antibody-induced bacteria 007j ~ expression FliC: j and fljA kind the gene deficient strain (Wt △ fljA) express FljB: z66 FliC: j. (2) PCR and sequence analysis confirmed successful build expression vector pBADfljA, and the expression of FljA fusion protein induced with 0.2% L-arabinose. Empty vector pBAD / g Ⅲ and expression vectors pBADfljA import 007j ~, by Western immune blotting found containing empty vector 007j to 007j ~ bacteria of FliC: j expression did not differ significantly, while contain the expression vector 007j ~ bacteria almost do not express FliC: j. (3) by real-time quantitative RT-PCR was found 007j ~ strains containing the expression vector fliC mRNA than 007j to strain reduced about 7 times. (4) PCR and sequence analysis confirmed the fliC gene mutant middle part of 3550bp fragment of lacZ alternative j antigen fliC gene lacZ insertion mutant 007j to (fliC :: lacZ) was successfully constructed. (5) Determination of β-galactosidase was found to contain the expression vector 007j ~ (fliC :: lacZ) mutant of the β-galactosidase enzyme activity about 100 times lower than 007j ~ (fliC :: lacZ) mutant . The conclusion: the Salmonella typhi GIFU10007 linear plasmid-like gene on the fljA Ⅰ phase whip Mao Suji the fliC suppression gene, and may be in the post-transcriptional regulation the fliC gene's expression.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria > Enteric bacilli > Salmonella typhi
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