CAJ | 학술논문

目的：筛选布鲁氏菌侵染牛胚胎滋养层细胞过程中与Ⅳ型分泌系统VirB4蛋白结合的潜在靶蛋白。方法设计引物并PCR扩增布鲁氏菌的virB4基因，构建表达载体pGBKT7-VirB4，酶切鉴定，测序分析正确后，转化酿酒酵母菌感受态细胞Y187，进行自激活和毒性检测；建立布鲁氏菌侵染牛胚胎滋养层细胞模型，构建布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库；采用酵母双杂交技术筛选与VirB4相互作用的滋养层细胞蛋白，实时定量PCR检测靶蛋白表达量的变化。结果成功构建了pGBKT7-virB4诱饵质粒，转入Y187后无毒性，不能自激活；获得了布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库；筛选到了13个阳性质粒，其中蛋白辅酶Q10和SLC3A2在布鲁氏菌侵染后mRNA表达量均增加。结论本试验对VirB4蛋白与宿主细胞的互作研究为进一步阐明布鲁氏菌感染宿主细胞的发病机制奠定了基础。
목적：사선포로씨균침염우배태자양층세포과정중여Ⅳ형분비계통VirB4단백결합적잠재파단백。방법설계인물병PCR확증포로씨균적virB4기인，구건표체재체pGBKT7-VirB4，매절감정，측서분석정학후，전화양주효모균감수태세포Y187，진행자격활화독성검측；건립포로씨균침염우배태자양층세포모형，구건포로씨균침염우배태자양층세포cDNA문고；채용효모쌍잡교기술사선여VirB4상호작용적자양층세포단백，실시정량PCR검측파단백표체량적변화。결과성공구건료pGBKT7-virB4유이질립，전입Y187후무독성，불능자격활；획득료포로씨균침염우배태자양층세포cDNA문고；사선도료13개양성질립，기중단백보매Q10화SLC3A2재포로씨균침염후mRNA표체량균증가。결론본시험대VirB4단백여숙주세포적호작연구위진일보천명포로씨균감염숙주세포적발병궤제전정료기출。
Potential target proteins binding to VirB4 of type Ⅳ secretion system were screened during Brucella infected bovine embryonic trophoblast cells .Brucella VirB4 genes were amplified by PCR with species-specific primers .Expression vector pGBKT7-virB4 was constructed and analysed by sequencing and restriction enzymes ,transforming to the yeast strain Y187 and testing self-activation and toxicity .The cells model and cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain were constructed respectively .Utilizing yeast two-hybrid system was employed to screen the target proteins of bovine embryo trophoblastic cells which was conjunctive with virB4 .These proteins were detected by real-time fluo-rescence quantitative PCR .The results suggested that bait plasmid pGBKT7-virB4 was successfully transformed into the Y187 and there was no toxicity and self-activation;the cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain was constructed .There screened 13 positive plasmids in which Q10 and SLC3A2 were up-regulated at the mRNA level .In this paper ,we reported the interactions between the VirB4 protein of Brucella and the bovine embryo trophoblastic cells ,which provide an upstream work for further elucidating the pathogenesis of Brucella infection of the host cell .