SS2三种毒力相关蛋白的克

发布时间：2018-03-18 18:02

本文选题：SS2　切入点：MRP　出处：《南京农业大学》2006年硕士论文　论文类型：学位论文

【摘要】：猪链球菌2型(Streptococcus suis 2，SS2)可以导致猪脑膜炎、关节炎、败血症、浆膜炎、心内膜炎、多发性关节炎等，对从业人员带来严重的威胁，可以导致人的脑膜炎、心内膜炎及中毒性休克综合症等严重疾患。目前国内外对SS的研究主要集中在SS2毒力相关因子上，目前公认的毒力相关因子有溶菌酶释放蛋白(muramidasereleased protein, MRP)、胞外蛋白因子(extracellular factor protein, EF)、溶血素(suilysin,本文简称sly)、荚膜多糖(capsular polysaccharide, CPS)等。由于对SS2毒力因子的了解较少限制了SS疫苗的研究及猪链球菌病的治疗与控制。本试验对1998年江苏海安病人分离株Habb MRP进行研究；对2005年四川资阳病人分离株分别进行EF和sly的研究。制备了MRP及EF单克隆抗体，并摸索MRP ELISA检测方法。克隆及表达SS2人源分离株Habb mrp基因片段，构建原核表达质粒pGEX4T-2-mrp。在大肠杆菌中诱导带有谷胱甘肽转移酶(GST)标签的融合蛋白MRP-GST表达；经亲和层析法纯化后，用凝血酶酶切去除重组蛋白中的GST，制备纯化的MRP抗原。序列分析表明，获得的mrp基因长957bp；原核表达的融合蛋白MRP-GST的分子量约61kD；凝血酶处理的MRP抗原的分子量约为35kD。Western blot分析显示，MRP-GST、MRP蛋白均可与MRP多克隆抗血清发生特异性反应。成功地克隆人源分离株Habb的mrp基因片段，并在原核系统实现高效表达，制备的纯化抗原MRP具有良好的抗原性，为开展相关免疫学研究奠定了基础。 SS2 MRP单克隆抗体的研制及其生物学特性的鉴定。以纯化的MRP-GST蛋白为免疫原，常规免疫BALB／c小鼠，采用B淋巴细胞杂交瘤技术进行细胞融合，并以经凝血酶酶切去除GST标签的MRP筛选细胞，，经反复筛选和多次克隆化培养，筛选出特异分泌鼠抗MRP单克隆抗体的杂交瘤细胞株。采用Western blot对单抗进行生物学特性的鉴定。成功获得6株持续、稳定分泌鼠抗MRP单克隆抗体的杂交瘤细胞株，命名为1G1、1B6、288、3G9、2C3及3E5。用表达纯化的MRP抗原、HRP标记的MRP抗原和一株MRP单抗腹水、以及ZY05719全菌兔多抗血清及猪多抗血清，建立检测猪链球菌MRP抗原及SS MRP抗体的ELISA方法。SS MRP抗血清的检测采用纯化的MRP抗原或灭活ZY05719细菌
[Abstract]:Streptococcus suis 2 SS2) can cause meningitis, arthritis, septicemia, serositis, endocarditis, polyarthritis, etc. Serious diseases such as endocarditis and toxic shock syndrome. At present, the research on SS mainly focuses on the virulence related factors of SS2. The commonly recognized virulence related factors are lysozyme releasing protein (MRP), extracellular protein (factor), extracellular protein (EFS), hemolysin (Slycerin), capsular polysaccharide (CPSs) and so on. The knowledge of SS2 virulence factors has limited the availability of SS vaccine. Study on the treatment and control of Streptococcus suis. In 1998, the Habb MRP isolated from Haian patients in Jiangsu Province was studied. In 2005, EF and sly of Ziyang patient isolate were studied respectively. Monoclonal antibodies against MRP and EF were prepared, and MRP ELISA detection method was explored. The Habb mrp gene fragment of SS2 human isolate was cloned and expressed, and the prokaryotic expression plasmid pGEX4T-2-mrp. the fusion protein MRP-GST with glutathione transferase tag was induced in Escherichia coli and purified by affinity chromatography. The purified MRP antigen was prepared by digesting GSTs from recombinant protein by thrombin digestion. The length of mrp gene was 957 BP, the molecular weight of prokaryotic expression fusion protein MRP-GST was about 61kD. the molecular weight of thrombin treated MRP antigen was about 35kD. Western blot analysis showed that MRP-GST-MRP protein could react specifically with MRP polyclonal antiserum. The mrp gene fragment of Habb isolated from Longren strain. The purified antigen MRP was highly expressed in prokaryotic system and had good antigenicity, which laid a foundation for the study of immunology. Preparation of monoclonal antibody to SS2 MRP and identification of its biological characteristics. The purified MRP-GST protein was used as immunogen to routinely immunize BALB/c mice, and B lymphocyte hybridoma technique was used for cell fusion. MRP, which was digested by thrombin to remove the GST label, was used to screen the cells. The hybridoma cell lines secreting murine monoclonal antibody against MRP were screened. Six hybridoma cell lines, which secreted murine anti-#en3# monoclonal antibody stably and continuously, were successfully obtained by Western blot. The hybridoma cell lines were named 1G1B6C28883G9O2C3 and 3E5. HRP-labeled MRP antigens and a MRP monoclonal antibody ascites were used to express purified MRP antigens, as well as ZY05719 whole bacterial rabbit polyantibodies and porcine polyclonal antisera. Establishment of ELISA method for Detection of MRP Antigen and SS MRP Antibody of Streptococcus suis. Detection of SS MRP Antiserum using purified MRP Antigen or inactivated ZY05719 bacteria
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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