【作者】 张辉；

【导师】 陈创夫；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 布鲁氏菌病(简称布病)是由布鲁氏菌引起的一类动物源性人兽共患病,在世界范围内广泛流行,尤其是发展中国家。近几年回升势头迅猛,引起了世界范围的广泛关注,为此,我国紧急发布了《关于加强布鲁氏菌防治工作的通知》。布鲁氏菌是一种兼性胞内寄生致病菌,侵袭力强、传染途径多,人感染后呈持续性感染,目前尚无法根治；家畜感染后主要引发母畜流产,给畜牧业生产造成严重损失。动物胎盘中含有大量的赤藓醇,能够促进布鲁氏菌的生长,布鲁氏菌对胎盘具有显著的亲嗜性,胚胎滋养细胞是母体和胎儿的联系纽带,同时也是布鲁氏菌感染的靶细胞,一旦受到损伤,就会导致流产,但引发流产的分子机制还不清楚。为此,本研究以布鲁氏菌和胚胎滋养层细胞为研究对象,主要展开以下的研究工作：1.羊种布鲁氏菌的分离鉴定及ELISA检测方法的建立。通过采用细菌群体形态观察、PCR、生化试验对分离培养的菌株进行鉴定,获得了羊种布鲁氏菌生物3型1株,命名为027株,对其SP41基因进行克隆、表达、纯化,包被酶标反应板并进行样本检测。结果布鲁氏菌病间接ELISA检测方法的最优条件是重组蛋白SP41最佳抗原包被浓度为0.6μg/mL,最佳血清稀释度为1：50,该方法重复性好、特异性强,样本阳性检出率高于虎红平板试验和试管凝集试验。2.羊种布鲁氏菌ery操纵子在感染人胚胎滋养层细胞HPT-8过程中的表达调控。采用实时定量PCR方法检测了布鲁氏菌侵染HPT-8细胞过程中ery操纵子4个基因在未侵染、侵染20min、1h、2h、3h、4h时的表达差异,结果ery操纵子在侵染前后的表达有明显差异,eryA、eryB、eryC相对表达量在侵染2h时表达量最高,eryD在3h时的相对表达量最高,其余状态下的表达量都很低,并且随着eryD表达量升高,eryA、eryB、eryC的表达量降低。3.建立羊种布鲁氏菌027株侵染HPT-8细胞的cDNA文库。分别提取布鲁氏菌侵染20min、1h、2h、3h、4h后HPT-8细胞总RNA,逆转录合成cDNA,同源重组法构建布鲁氏菌侵染HPT-8细胞的cDNA文库,并对部分EST进行测序分析,结果羊种布鲁氏菌027株侵染人滋养层cDNA文库的库容为1.43×106,重组率为96.92%,插入片段大小在0.2-5kb；对文库63个克隆进行测序,用BlastX和BlastN进行序列同源性比对,并将63个基因进行了功能分类。然后,构建pGBKT7-omp25诱饵质粒,转化酵母Y187,结果表明诱饵菌无自激活活性、无毒性。4.筛选并验证与布鲁氏菌流产相关因子互作的HPT-8细胞靶蛋白。采用酵母双杂交技术筛选与布鲁氏菌外膜蛋白OMP25互作的HPT-8细胞捕获蛋白,并采用免疫共沉淀技术验证互作的蛋白。共筛选出了7个与布鲁氏菌OMP25诱饵蛋白相结合阳性捕获蛋白,验证试验表明布鲁氏菌外膜蛋白OMP25与HPT-8细胞的Ferritin、hypothetical LOC339123蛋白发生了相互作用。5.分析捕获蛋白在布鲁氏菌侵染HPT-8细胞过程中的作用。根据铁蛋白、hypothetical LOC339123蛋白核苷酸序列,分别构建了3个特异性shRNA干扰载体,转染HPT-8细胞,实时定量PCR检测2个基因的表达水平,检验其干扰效率；采用布鲁氏菌感染转染shRNA干扰载体前、后的HPT-8细胞,检测布鲁氏菌的相对数量。结果针对铁蛋白、hypothetical LOC339123合成的shRNA干扰片段与各自的混合组的干扰效果基本一致,Ferritin混合组的干扰抑制率为76.18%, hypothetical LOC339123混合组的干扰抑制率为71.11%。针对铁蛋白的干扰载体混合组转染前后,布鲁氏菌相对数量差异较大,而hypotheticalLOC339123的差异不明显。以上研究结果表明,分离培养的布鲁氏菌027株为羊种布鲁氏菌生物3型；成功构建了基于重组蛋白SP41的布鲁氏菌病间接ELISA检测方法；布鲁氏菌感染HPT-8细胞过程中ery操纵子4个基因在不同时间点表达存在着差异；成功构建了羊种布鲁氏菌027株侵染HPT-8细胞的cDNA文库；布鲁氏菌OMP25蛋白与HPT-8细胞的Ferritin、hypothetical LOC339123发生了相互作用,Ferritin与布鲁氏菌感染相关,hypothetical LOC339123与布鲁氏菌感染不相关。通过本研究,为揭示布鲁氏菌引发流产的分子机制提供线索,也为布鲁氏菌病新药物研发、防治及家畜抗病育种工作奠定基础。更多还原

【Abstract】 Brucellosis is a zoonotic disease caused by members of the genus Brucella. It is widely popular in the world, especially in developing countries. Recently, the rapid increasing cases in human and animals have caused widespread concern around the world. China released an emergent document "on the strengthening of prevention and treatment of Brucellosis notice". Brucella is a facultative intracellular parasitic bacterium. Due to its strong invasion and multiple approaches of transmission, patients show persistent infection, which is usually known as incurable disease. Infectious animal mainly caused abortion, which leads to serious loss for livestock industry. Animal placenta contains much Erythritol, which can promote the growth of Brucella. Embryonic trophoblast, knows as target cell of brucella, is a link between the pregnant animal and the fetus. Once infected, it will lead to abortion. However, its molecular mechanism of abortion is unclear. In this study, based on Brucella and embryonic trophoblast cells, the main research work carried out as the follows:1. Isolation and Identification of Brucella melitensis and ELISA method were constructed. According to bacterial morphology, PCR, biochemical examination, Brucella melitensis type 3, named as 027 strain, was obtained. After its SP41 gene was cloned, expressed, purified, indirect ELISA method was built. During our experiments, the best concentration of SP41 recombinant protein is 0.6μg/mL, the best dilution of serum is 1:50. The method has good reproducibility, specificity, positive rate of samples was higher than the rose Bengal plate test (RBPT) and standard tube agglutination test(SAT).2. Study on ery operon expression and regulation of Brucella melitensis during human embryonic trophoblast HPT-8 infected with pathogen. The real-time quantitative PCR method was used to detect expression differences of ery operon 4 genes in non-infected, infected 20min, 1h,2h,3h, and 4h. ery A, eryB and eryC in 2h has a significant over-expression. eryD in 3h has the highest expression compared to ery A, eryB and eryC. Interestingly, with the increase of eryD expression, ery A, eiyB and eryC decreased.3. The cDNA library of HPT-8 cells infected with Brucella melitensis 027 strain was constructed. The total RNA of infected cells at 20min, 1h,2h,3h and 4h Were extracted respectively. With cDNA synthesized by reverse transcriptase, homologous recombination construct infected HPT-8 cell cDNA library. The partial ESTs were sequenced. The results showed that cDNA library was successfully constructed. cDNA capacity was 1.43×106, the recombinant rate was 96.92 percent. The size of the inserted fragments is between 0.2-5kb.63 clones of cDNA library were random selected and sequenced. These gene were classified by BlastX and BlastN. The bait plasmid pGBKT7-omp25 was constructed into yeast Y187, which results show that the bait yeast is non-self-activated and non-toxic.4. The target protein were screened and verified. OMP25 interacted with 7 positive protein of HPT-8 cells by yeast two-hybrid. Ferritin and hypothetical LOC339123 were verified by immunoprecipitation.5. Study on the function of prey protein. According to nucleotide sequences of ferritin and hypothetical LOC339123, three specific interfering shRNA vectors transfecting HPT-8 cells, respectively, real-time quantitative PCR detected. Ferritin mixed group interference suppression rate of 76.18%, hypothetical LOC339123 mixed group interference suppression rate of 71.11%. After Ferritin was interfered, the relative number of Brucella in HPT-8 cells increased compared to hypothetical LOC339123 protein.The above results show that the isolate was Brucella melitensis type 3; indirect ELISA based on SP41 was successfully built; Brucella ery operon 4 genes expression differed at different times during infecting HPT-8 cells; The cDNA library of HPT-8 cells infected with Brucella melitensis 027 strain was successfully constructed; Brucella OMP25 interacted with HPT-8 Cell’s ferritin and hypothetical LOC339123 protein; ferritin is related to Brucella infection while hypothetical LOC339123 protein is not. In this study, It is useful to demonstrate molecular mechanism of abortion, to screen specific candidate drugs to brucellosis, to give a upstream work for breeding of anti-brucellosis livestock.更多还原