【作者】 孙瑜隆；

【导师】 魏虎来； 柳纪省；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 猪链球菌(Streptococcus suis S.suis)是一种重要的人畜共患病原体，可引起猪脑膜炎、心内膜炎、关节炎及败血症等疾病，亦可引起人类感染，导致脑膜炎及中毒性休克综合征等严重疾患。该菌有33个血清型，其中以2型流行最广、致病性最强。猪链球菌有多种毒力因子，如溶菌酶释放蛋白(MRP)，溶血素(SYL)，胞外蛋白因子(EF)等，但MRP是该菌最为重要的毒力因子之一。本研究根据猪2型链球菌溶菌酶释放蛋白编码基因序列，设计并合成1对特异性引物，以猪2型链球菌青海株基因组为模板扩增MRP基因34-813bp片断，并进行T/A克隆，转化大肠杆菌，成功获得目的片断，将目的片段定向克隆到pET-28a(+)组氨酸的下游。用鉴定阳性的重组质粒转化BL21，经IPTG诱导可表达分子量约27kD的融合蛋白。免疫印迹分析表明，该融合蛋白具有MRP的抗原表位。本研究克隆了猪2型链球菌青海株MRP基因完整的阅读框。编码区长度为3771bp，与已登陆的序列对比发现此基因的阅读框高度保守。将克隆的青海株的MRP基因插入pAdTrack-CMV穿梭质粒，获得了转移质粒pAdTrack-CMV/MRP，对之线性化后，用pAdEasyTM系统，电转化已含有腺病毒骨架载体pAdEasy-1的感受态大肠杆菌，经同源重组，获得含有全MRP基因的腺病毒载体pAd/MRP，后者经酶切充分暴露反向末端重复序列后，与脂质体混合转染HEK-293细胞，获得了复制缺陷型重组腺病毒rAd-MRP。RT-PCR检测到重组腺病毒rAd-MRP中MRP基因在转录水平有表达。连续传代和PCR检测表明rAd-MRP在HEK-293细胞中传代稳定。为评价重组腺病毒rAd-MRP的免疫效果，用其免疫28日龄小鼠后，间接ELISA可以检测到特异性抗体，抗体滴度达1：160。更多还原

【Abstract】 Streptococcus suis (S.suis) is an important swine pathogen that cause a number of pathological diseases, such as meningitis, endocarditis, arthritis and septicemia. There are 33 serotypes of S.suis, among which type 2 is the most popular and pathogenic. S.suis type 2 produce such virulence factors as muramidase-released protein (MRP), suilysin, extracellular factor (EF), etc. but MRP is one of the most important ones.The gene fragment of 34 to 813 bp (780bp) in length encoding the muramidase-released proteins was amplified from genomic DNA of Streptococcus suis type 2 strain QINGHAI by PCR. The PCR products was later cloned into T vector and transformed into host strain E.coli, and the plasmid DNA of positive clones was extracted, digested with enzymes and purified. Then the fragment was cloned into expression vector PET-28a(+). The recombinant plasmid was transformed into E.coli BL21. The fusion protein with a molecular weight of about 27KD could be expressed after induction with IPTG. and it possesses antigenic epitopes of muramidase-released protein.The gene encoding MRP of S.suis type 2 was cloned and subcloned into shuttle vector pAdTrack-CMV, forming transfer vector of pAdTrack-CMV/MRP, Linearized pAdTrack-CMV/MRP was electrotransformed into E. coli strain BJ5183 which is RecA+ and perform homologous recombination with supercoiled adenoviral vector pAdEasy-1 which contained in BJ5183 cells, the resultant recombinant which exposed inverted terminal repeat (ITR) sequence by restriction enzyme digestion was transfected into HEKAD-293 with lipoplast to produce the recombinant adenovirus (rAd-MRP). RT-PCR was used to detect the transcription of MRP, and serial passage and PCR were used to detect the stability of rAd-MRP. To evaluate the immune activity, rAd-MRP was inoculated to 28-day-old mouses by muscular injection and abdominal injection respectively, indirect enzyme linked immunosorbent assay (ELISA) was used to detect the titres of MRP specific antibody. the antibody titres reached 1:160.更多还原