流产布鲁氏菌omp25基因的克隆与原核表达及表达产物免疫反应性的测定
发布时间：2019-06-18 20:34
【摘要】：布鲁氏菌病(Brucellosis)是由布鲁氏菌(Brucella. spp)引起的人、畜共患传染病。本病广泛分布于世界各地,给畜牧业和人类的健康带来严重危害。流产布鲁氏菌(B. abortus)是兼性细胞内寄生菌,主要宿主是牛,也可感染人,引起波浪热。目前,有些国家使用弱毒疫苗预防本病,能够起到一定的免疫效果,但弱毒疫苗存在许多潜在问题。因此布鲁氏菌病的诊断和防制成为该病研究中的热点。研究和开发布鲁氏菌病诊断技术和新型疫苗,将对我国控制和根除布鲁氏菌病具有重要意义。 本实验利用聚合酶链式反应(PCR)从流产布鲁氏菌基因组DNA中扩增出外膜蛋白基因omp25(outer membrane protein 25Kda),测序证实所克隆的基因与国外报道的流产布鲁氏菌omp25基因的核苷酸序列同源性为100%,推测的氨基酸序列同源性为100%;且与布鲁氏菌其他种的同源性也高达98%以上。将omp25克隆至载体pGEX-6P-1,转化于大肠杆菌BL21(DE3)中表达获得GST融合蛋白,SDS-PAGE分析证明,表达产物为51ku的融合蛋白,以包涵体的形式存在。免疫学分析(Western blotting、ELISA)表明,所表达的蛋白可以与流产布鲁氏菌阳性牛血清反应;将表达产物电泳后切胶回收进行间接血凝试验(IHA),结果表明所表达的融合蛋白能够引起流产布鲁氏菌阳性牛血清发生凝集反应,具有免疫反应性。应用蛋白质专家系统对Omp25蛋白进行结构和功能预测,发现Omp25在14～35个氨基酸处有一个信号肽识别位点;预测结果还表明Omp25蛋白在6～23位氨基酸处有一个跨膜螺旋区。
[Abstract]:Brucellosis (Brucellosis) is caused by brucellosis (Brucella.) Spp) caused by zoonotic infectious diseases. The disease is widely distributed all over the world and brings serious harm to animal husbandry and human health. Brucella aborted (B. abortus) is a facultative intracellular parasite, the main host is cattle, can also infect people, causing wave fever. At present, some countries use attenuated vaccine to prevent this disease, which can play a certain immune effect, but there are many potential problems of attenuated vaccine. Therefore, the diagnosis and control of brucellosis has become a hot spot in the study of brucellosis. The research and development of diagnostic techniques and new vaccines for brucellosis will be of great significance to the control and eradication of brucellosis in China. In this experiment, the outer membrane protein gene omp25 (outer membrane protein 25Kda was amplified from the genomic DNA of Brucella aborted by polymerase chain reaction (PCR). Sequencing confirmed that the homology of the cloned gene with the omp25 gene of Brucella aborted was 100%, the deduced amino acid sequence homology was 100%, and the homology with other species of Brucella was more than 98%. Omp25 was cloned into vector pGEX-6P-1, and transformed into E. coli BL21 (DE3) to express GST fusion protein. SDS-PAGE analysis showed that the expressed product was 51ku fusion protein, which existed in the form of inclusion body. Immunological analysis (Western blotting,ELISA) showed that the expressed protein could react with Brucella aborted positive bovine serum, and the expressed product was recovered by gel cutting and indirect hemagglutination test (IHA), results showed that the expressed fusion protein could cause the aggregation reaction of Brucella aborted bovine serum and had immunoreactivity. The structure and function of Omp25 protein were predicted by protein expert system, and it was found that Omp25 had a signal peptide recognition site at 14 鈮，
本文编号：2501772
[Abstract]:Brucellosis (Brucellosis) is caused by brucellosis (Brucella.) Spp) caused by zoonotic infectious diseases. The disease is widely distributed all over the world and brings serious harm to animal husbandry and human health. Brucella aborted (B. abortus) is a facultative intracellular parasite, the main host is cattle, can also infect people, causing wave fever. At present, some countries use attenuated vaccine to prevent this disease, which can play a certain immune effect, but there are many potential problems of attenuated vaccine. Therefore, the diagnosis and control of brucellosis has become a hot spot in the study of brucellosis. The research and development of diagnostic techniques and new vaccines for brucellosis will be of great significance to the control and eradication of brucellosis in China. In this experiment, the outer membrane protein gene omp25 (outer membrane protein 25Kda was amplified from the genomic DNA of Brucella aborted by polymerase chain reaction (PCR). Sequencing confirmed that the homology of the cloned gene with the omp25 gene of Brucella aborted was 100%, the deduced amino acid sequence homology was 100%, and the homology with other species of Brucella was more than 98%. Omp25 was cloned into vector pGEX-6P-1, and transformed into E. coli BL21 (DE3) to express GST fusion protein. SDS-PAGE analysis showed that the expressed product was 51ku fusion protein, which existed in the form of inclusion body. Immunological analysis (Western blotting,ELISA) showed that the expressed protein could react with Brucella aborted positive bovine serum, and the expressed product was recovered by gel cutting and indirect hemagglutination test (IHA), results showed that the expressed fusion protein could cause the aggregation reaction of Brucella aborted bovine serum and had immunoreactivity. The structure and function of Omp25 protein were predicted by protein expert system, and it was found that Omp25 had a signal peptide recognition site at 14 鈮，
本文编号：2501772
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