摘要
本研究旨在研究布鲁菌糖基转移酶(WboA)在诱发胚胎滋养层细胞(HTP-8)炎症反应中的作用。以羊种布鲁菌M5-90为模板,扩增WboA基因,构建pET28a(+)-WboA重组表达质粒,转化重组质粒在BL21(DE3)中表达,通过SDS-PAGE和Western blot鉴定,并对重组WboA蛋白进行纯化。构建自杀载体pGEM-7zf-ΔWboA-SacB,通过同源重组的方法,筛选布鲁菌WboA基因缺失株ΔWboA,并进行PCR鉴定和遗传稳定性检测。然后将纯化的WboA蛋白与ΔWboA分别作用于胚胎滋养层细胞,ELISA检测IL-6、IL-10、TNF-α和乳酸脱氢酶(LDH)的相对变化量。结果成功纯化了WboA蛋白,构建了ΔWboA,ΔWboA侵染胚胎滋养层细胞诱导产生炎症细胞因子IL-6、IL-10和TNF-α均低于M5-90对照组,差异显著(P<0.05),WboA蛋白作用胚胎滋养层细胞时,炎症细胞因子IL-6、TNF-α和LDH均高于PBS对照组,差异极显著(P<0.01)。本研究表明WboA蛋白具有细胞毒作用,布鲁菌的致炎作用与脂多糖O链相关,为进一步阐明布鲁菌感染宿主细胞的发病机制奠定了基础。
The study explored the role of glycosyltransferase in inflammation response of embryo trophoblast cells infection by Brucella. WhoA gene was amplified from Brucella melitensis M5-90 strain by PCR and cloned into vector pET-28a（＋）. The constructed recombinant plasmid pET WhoA was transformed to competent E. coli BL21. The protein Whoa was identified by SDS- PAGE, Western blot, and purified. The suicide plasmid pGEM-7zf-AWboA SacB was construc- ted. The WhoA gene was knocked out of the genomic DNA of Brucella melitensis vaccine M5-90 strain by homologous recombination. Then purified the WboA protein and △WboA affects in the embryo trophoblast cells separately, the inflammatory cytokines of IL-6, IL-10, TNF-α and the lactic dehydrogenase （LDH） were ELISA examination. The protein WboA was successfully puri lied and the mutant AWboA was constructed. Compared with the M5-90 infected group, the HPT-8 cells infected by AWboA released much less inflammatory cytokine IL-6, IL-10 and TNF-α （P〈0. 05）. The inflammatory cytokine IL-6, TNF-α and LDH released by HPT-8 effected with WboA protein were significantly higher than the PBS control group（P〈0.01）. These re sults indicate that the protein WboA has the function of cytotoxin and the inflammation response of Brucella is related to the Lipopolysaccharides O chain.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2012年第9期1437-1443,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家“973项目”(2010CB30203)
国家自然科学基金(31001046/30960288)
高层次人才科研启动资金(RCZX200914)
