Study On The Immunity And Eukaryotic Expression Of The Recombinant Gene Of Leukotoxin From The Pathogenic Fusobacterium Necrophorum

This study objective gene, which were amplification from extraction DNA fromdomestic isolating pathogenic bovine Fusobacterium necrophorum(F.n.) F4, constructedefficient eukaryotic expression vector by PCR and cloning technology, induction expressionwith Pichia pastoris. In addition, the immunological activity and cytotoxic characteristicsof the recombination protein were studied respectively in vitro. Contents are as follows:According to the literature and bioinformatics technology, four pairs of primers weredesigned refer to the lkt(AF312861)of the standard F.n. strain reported at GenBank. Thebsbse, gas-sh and sh of lkt were amplified by PCR, which were constructed the bsbse,gas-sh and sh TA clone vector, then sequencing and submitting. The bsbse-gas-sh andbsbse-sh were obtained using the T4DNA Ligase. Construction eukaryon expression vectorpPIC9K-bsbse-sh and pPIC9K-bsbse-gas-sh, and were transformed to protoplast KM71Hafter linearization. Two secretion expression eukaryon engineering bacterias were to obtain,by His-auxotrophic and G418culture medium screening and PCR identification. Under theinduction of1.0%methanol for4d, the BSBSE-SH and BSBSE-GAS-SH fusion proteinwas highly expressed into supernatant. With the analysis of SDS-PAGE, the molecularweight of BSBSE-SH and BSBSE-GAS-SH were53.2KDa and117.9KDa respectively.The recombination protein were analysised through the Western Blot, the suggestivefrom results that they have the immunogenicity. There are several blots bigger thancomputed value53.2KD, they are supposed polymers of the recombination proteinBSBSE-SH, the blot of the recombination protein BSBSE-GAS-SH is obviously, presumethat the hydrophobicity fragment GAS have stabilization and repellent effects when the threedimensional structures of the recombination protein BSBSE-GAS-SH is to form. Using NCTCclone1469and Ana-1cells, that did MTS dye reduction assay in vitro. The results showedthat the recombination protein BSBSE-SH and BSBSE-GAS-SH of F.n. LktA coulddemonstrate cytotoxic effects on host cells. The BSBSE-SH was more cytotoxic effects afterdilution by cell culture medium. The high concentration of the recombination protein caninduce apoptosis directly, the low concentration can stimulate resistance of cells. This studythat recombination proteins were efficient expression from leukotoxin of F.n. usingeukaryocyte expression vector, and has laid the basic data to prepare for purificationactiving pragment of LktA, construction, function and a new F.n. subunit vaccine.