The Express Of Truncated Gene And Original Sexual Response Analysis For Cattle Footrot Using Fusobacterium Necrophorum Leukotoxin, Haemolysin And43K OMP

The major virulence factor of necrosis Bacillus, neutralizing antigen epitope area ofVirulence factors leukotoxin (lktA), hemolysin (hly) and43K outer membrane protein,the studybase on preparation of a multi neutralization antigenic protective antigen so as to providetheoretical basis for Prevention and treatment of necro bacillosis of cattle and sheep.Firstly, in preliminary studies, has obtained3main antigen region, PL1, PL3, PL4, ofFusobacterium necrophorum leukotoxin genes.Then, according to the full-length4107bp genesequence, Fusobacterium necrophorum hemolysin of necrosis Bacillus H05strain which waspublished in Genbank, the necrosis Bacillus hemolysin gene was truncated expression, and5pairs of hemolysin gene of Truncated Fragment containing BamHⅠand XhoⅠ were designed.five truncated overlapping fragments were amplifyed with PCR technique, PCR products werecloned to vector pET-32a, and The5positive plasmid with truncated fragment was transformedinto the competent cells of E.coli BL21. The E.coli BL21(DE3) containing recombinantplasmid, which screening with PCR technique, were induced by IPTG. Expression ofrecombinant protein were analysised antigen differentiation by Western blot. The Results showsthat,3of the5recombinant protein, hly-2(275-567aa), hly-3(566-888aa), hly-4(887-1163aa),were succeed expression with antigenicity. We carried out the following experiment conten inorder to accurate the epitope region, so as to eliminate the Non essential gene fragmente, wecarried out the following experimentcontent:1. According to hemolysin gene truncatedfragments of hly-2, hly-3, hly-4and necrophorum leukotoxin gene truncated fragments of PL1,PL3, PL4of necrosis Bacillus H05strain nucleotide sequence published in Genbank. The genesequence of43K Outer membrane protein With outer membrane protein of Fusobacteriumnuclea wereamplification, And the cloned genes are truncated to express. specific primers weredesigned containing BamHⅠ and XhoⅠ sites, which12of leukotoxin,10of hemolysin,4ofthe43K outer membrane protein,the truncated fragments wereamplified by PCR amplified byPCR truncated fragments of each geneoverlapping, and then the PCR products amplified byBamHⅠ and XhoⅠ restriction enzyme digestion were cloned into vector pET-32a;2. Thepositive recombinant plasmid with truncated fragments were transformationed into competentcells of E.coli BL21, using PCR method to screening positive colonies, were induced by IPTG, and the successful expressioned of recombinant protein were antigenicity analysis by Westernblot technology.The results showed that,10of the12truncated gene were successful expression ofleukotoxin,and the6of the recombinant protein with antigenicity, respectively PL3-1(1-103aain PL3), PL3-2(97-199aa in PL3), PL3-5(383-484aa in PL3), PL3-7(576-708aa in PL3),PL4-2(105-215aa in PL4); PL4-3(209-329aa in PL4);6of the10truncated fragments ofhemolysin gene were successful expression, the2of the recombinant protein with antigenicity,respectively H2(98-205aa in hly-2), H9(93-194aa in hly-4);4gene were successful expressionof the43K outer membrane protein, and the2of the recombinant protein with antigenicity,respectively43K-1(19-108aa),43K-2(101-199aa).