【摘要】：布魯氏菌病(Brucellosis)是由布魯氏菌(Brucella. spp)引起的人、畜共患傳染病。本病廣泛分布于世界各地,給畜牧業(yè)和人類的健康帶來嚴重危害。流產布魯氏菌(B. abortus)是兼性細胞內寄生菌,主要宿主是牛,也可感染人,引起波浪熱。目前,有些國家使用弱毒疫苗預防本病,能夠起到一定的免疫效果,但弱毒疫苗存在許多潛在問題。因此布魯氏菌病的診斷和防制成為該病研究中的熱點。研究和開發(fā)布魯氏菌病診斷技術和新型疫苗,將對我國控制和根除布魯氏菌病具有重要意義。 本實驗利用聚合酶鏈式反應(PCR)從流產布魯氏菌基因組DNA中擴增出外膜蛋白基因omp25(outer membrane protein 25Kda),測序證實所克隆的基因與國外報道的流產布魯氏菌omp25基因的核苷酸序列同源性為100%,推測的氨基酸序列同源性為100%;且與布魯氏菌其他種的同源性也高達98%以上。將omp25克隆至載體pGEX-6P-1,轉化于大腸桿菌BL21(DE3)中表達獲得GST融合蛋白,SDS-PAGE分析證明,表達產物為51ku的融合蛋白,以包涵體的形式存在。免疫學分析(Western blotting、ELISA)表明,所表達的蛋白可以與流產布魯氏菌陽性牛血清反應;將表達產物電泳后切膠回收進行間接血凝試驗(IHA),結果表明所表達的融合蛋白能夠引起流產布魯氏菌陽性牛血清發(fā)生凝集反應,具有免疫反應性。應用蛋白質專家系統(tǒng)對Omp25蛋白進行結構和功能預測,發(fā)現Omp25在14～35個氨基酸處有一個信號肽識別位點;預測結果還表明Omp25蛋白在6～23位氨基酸處有一個跨膜螺旋區(qū)。
[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，

本文編號：2501772