【作者】 周明光；

【导师】 金梅林； 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医， 2007， 硕士

【摘要】 猪链球菌(Streptococcus suis，S．suis)能引起猪脑膜炎、心内膜炎、关节炎、肺炎和败血症等，该菌有35个血清型，其中2型流行最广、且致病性最强，并能感染人，是一种重要的人畜共患病病原。但猪链球菌经常与其他一些病原如猪放线杆菌、猪丹毒杆菌和猪蓝耳病病毒等发生混合感染，给临床诊断和抗体水平的检测造成了很大困难。因此，本课题的研究目的：1建立以猪2型链球菌荚膜多糖为抗原的间接ELISA检测方法。2构建溶血素基因的原核表达质粒，并分析表达产物的免疫反应性，建立以溶血素为靶蛋白的猪链球菌间接血凝(IHA)抗体检测方法。3以上述两种方法对临床上送检血清进行检测，验证建立的两方法的符合率，并分析最近一年内我国猪2型链球菌的流行病学特点。猪2型链球菌能产生多种毒力因子，常见的有荚膜多糖(CPS)、溶菌酶释放蛋白(Muramidase-released protein，MRP)、胞外因子(Extracellular factor，EF)，溶血素(Suilysin，SLY)等，其中CPS是最早被证实的猪2型链球菌毒力因子，也是进行分型的依据，本试验用改良的kodana方法提取猪2型链球菌的荚膜多糖，并以此作为抗原建立间接ELISA抗体检测方法，对各方面条件进行优化，研制出猪2型链球菌间接ELISA抗体检测试剂盒。另外发病猪的猪2型链球菌中有相当高比例的菌株表现出溶血素活性，因而猪2型链球菌溶血素被认为是该菌主要的毒力因子。本研究根据猪2型链球菌溶血素基因序列设计一对特异性引物，采用PCR扩增猪2型链球菌LT-1分离株溶血素基因，将扩增产物克隆到pMD18-T质粒载体中，酶切和PCR鉴定后测序鉴定。结果显示，扩增的LT-1株溶血素基因长度为1494bp，编码497个氨基酸。将溶血素基因克隆入表达载体pET-28a中，提取重组质粒进行PCR和酶切鉴定，筛选阳性克隆，用SDS-PAGE电泳和Western blot进行溶血素的表达和鉴定。结果表明表达产物具有免疫反应性。然后以表达的溶血素蛋白为抗原致敏绵羊红细胞，并对各方面条件优化，建立猪链球菌间接血凝试验(IHA)。用间接ELISA方法检测从河南、安徽、江西、湖南、湖北、福建、广东、广西和北京等地送检的血清样品共3991份，结果表明阳性率分别为33.75％，41.6％等，各省血清样品阳性率从0％到41.6％。本研究建立的两种检测方法操作简单、特异性强、敏感性高并适用于猪链球菌的抗体检测和流行病学调查。更多还原

【Abstract】 Streptococcus suis is an important swine pathogen that causes a. number ofPathological conditions such as meningitis, endocarditis, arthritis, pneumonia andsepticemia. There are currently 35 seroypes of Streptococcus suis, among which type 2 isthe most popular and pathogenic. But Streptococcus suis type 2 often combined infectionwith some other etiological agents as Actinobacillus suis, Bacillus erysipelatous andPRRS virus, It results in tremendous difficulty by clinical diagnosis and antibodiesdetection. Thus the topic purpose of this study:1 To establish indirect ELISA detection method of Streptococcus suis type 2 usedcapsular bolysaccharides as antigen.2 To construct the prokaryotic expression vectors for suilysin gene and examine itsimmunoreactivity of expression product and establish indirect hemagglutination assay(IHA) detection method of Streptococcus suis used Suilysin protein as antigen.3 Detect clinical serums by two above-mentioned kinds of method to check thecoincidence of two kinds of method and analyze the epidemiology characteristic ofStreptococcus suis type 2 in lately one year in our courtry.Streptococcus suis type 2 can create many kinds of virulence factor, for examplecapsular bolysaccharides(CPS), Muramidase-released protein (MRP),Extracellular factor (EF), Suilysin (SLY) and so on. Among the total, capsularbolysaccharides is most early corroborative virulence factor of Streptococcus suis type 2,it is also the base of typing. This test extracts capsular bolysaccharides by modifiedkodana method to establish indirect ELISA detection method and optimizes the bestworking condition to manufacture indirect ELISA detection kit Otherwise, Streptococcussuis type 2 has a high proportional to display suilysin activity, so Suilysinis knownas the virulence factor. On the base of the suquence of suilysin gene, The suilysin gene ofStreptococcus suis type 2 isolated LT-1 was amplified by PCR from bacterial genomicDNA, cloned into pMD18-T vector and then sequenced. The sequencing result showedthat the PCR product was composed of 1494 nucleotides encoding a polypeptide of 497amino acids. Streptococcus suis suilysin gene was cloned into a prokaryotic expression plasmid pET-28a, and the recombinant plasmid was then identifid by PCR and restrictionenzyme digestion and screened the positive clones. Suilysin expression was analyzed bySDS-PAGE and Western blot. Suilysin was expressed in the recombinant strains andreactive to positive serum as shown by immnuno-blotting. The sheep erythrocyte(SE) wasallergized by the expressed SLY protein and optimized all round conditions, then theindirect hemagglutination assay was established of Streptococcus suis.To detect 3 991 shares serum products by indirect ELISA detection method inHenan, Anhui, Jiangxi, Hunan, Hubei, Fujian, Guangdong, Guangxi and Beijing, the rateof positive was 33.75 %, 41.6% and so on. The two kinds of method established bythis research operate simplely, specificity highly, sensitivity highly and refered todetecting antibody and epidemiological survey agaist Streptococcus suis.更多还原