布鲁菌糖基转移酶对细胞炎症反应的影响

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (9): 1437-1443.doi:

布鲁菌糖基转移酶对细胞炎症反应的影响

张豫，张辉^*，张艳，孟茹，王震，陈瑞花，张俊波，鲁芝子，陈创夫^*

（石河子大学动物科技学院新疆地方与民族高发病教育部重点实验室，石河子 832003）

出版日期:2012-09-25 发布日期:2012-09-25
通讯作者: 陈创夫，教授，E-mail:ccf-xb@163.com；张辉，副教授，E-mail: allanzhh@yahoo.com
作者简介:张豫（1985-）,女，新疆石河子人，硕士生，主要从事布鲁菌致病机制的研究，E-mail:llb@shzu.edu.cn，Tel:09932060217
基金资助:
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）

The Effect of Cellular Inflammatory Response by Glycosyltransferase of Brucella

ZHANG Yu, ZHANG Hui^*, ZHANG Yan, MENG Ru, WANG Zheng, CHEN Run-hua,
ZHANG Jun-bo, LU Zhi-zi, CHEN Chuang-fu^*

(Key Laboratory of Xinjiang Endemic and Ethnic Disease, College of Animal
Science &Technologe, Shihezi University, Shihezi 832003, China)

Online:2012-09-25 Published:2012-09-25

摘要/Abstract

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。

Abstract: The study explored the role of glycosyltransferase in inflammation response of embryo trophoblast cells infection by Brucella. WboA gene was amplified from Brucella melitensis M5-90 strain by PCR and cloned into vector pET-28a(+). The constructed recombinant plasmid pET-WboA was transformed to competent E. coli BL21. The protein WboA was identified by SDS-PAGE, Western blot, and purified. The suicide plasmid pGEM-7zf-ΔWboA-SacB was constructed. The WboA gene was knocked out of the genomic DNA of Brucella melitensis vaccine M5-90 strain by homologous recombination. Then purified the WboA protein and ΔWboA 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 purified and the mutant ΔWboA was constructed. Compared with the M5-90 infected group, the HPT-8 cells infected by ΔWboA 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 results indicate that the protein WboA has the function of cytotoxin and the inflammation response of Brucella is related to the Lipopolysaccharides O chain.

中图分类号:

S852.614

张豫, 张辉, 张艳, 孟茹, 王震, 陈瑞花, 张俊波, 鲁芝子, 陈创夫. 布鲁菌糖基转移酶对细胞炎症反应的影响[J]. 畜牧兽医学报, 2012, 43(9): 1437-1443.

ZHANG Yu, ZHANG Hui, ZHANG Yan, MENG Ru, WANG Zhen, CHEN Rui-Hua, ZHANG Jun-Bo, LU Zhi-Zi, CHEN Chuang-Fu. The Effect of Cellular Inflammatory Response by Glycosyltransferase of Brucella[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(9): 1437-1443.

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