【作者】 张炜；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪链球菌(Streptococcus suis)是一种革兰氏阳性菌,有35个血清型,其中SS2致病力最强,可引致猪的急性败血症及相关人员感染而死亡,造成较大的经济损失,产生重大的社会影响,成为公共卫生突发事件,倍受世人关注。为了进一步了解SS2免疫及致病机制,本文使用蛋白组学的方法对其进行研究,以期发现新的免疫原性蛋白及寻找新毒力标志物。为了高通量地筛选亚单位疫苗的候选物,本试验采用了免疫蛋白组学的方法,对SS2的可刺激宿主产生抗体的分泌蛋白(extracellular proteins)、膜相关蛋白(membrane-associated proteins,MAP)和细胞壁蛋白(cell-wallproteins,CWP)分别进行了筛选。应用免疫蛋白组学方法,分析SS2的分泌蛋白,以SPF微型猪SS2康复血清为一抗,HRP-SPA为二抗,对转移到PVDF膜的中国分离株ZY05719及HA9801的上清蛋白,检测具有免疫原性的蛋白。将重复胶上与之对应的蛋白点进行MALDI-TOF-MS分析。共鉴定出7个免疫原性蛋白,其中包括溶菌酶释放蛋白(muramidase-released protein,MRP)、胞外因子(extracellurlar factors,EF)和溶血素(suilysin,SLY),同时还发现4个以前未被报道的免疫原性蛋白。用Triton X-114法富集SS2 HA9801的膜相关蛋白。作免疫蛋白组学分析,以SPF微型猪SS2免疫血清为一抗,HRP标记的羊抗猪为为二抗,对转移到PVDF膜上的HA9801膜相关蛋白进行检测。总共发现13个免疫原性的蛋白,将重复胶上相应的点进行MALDI-TOF-MS检测,全部成功地被鉴定出来,其中7个为首次报道。用变溶菌素法提取SS2 HA9801的细胞壁蛋白。应用免疫蛋白组法,以SPF微型猪的免疫血清为一抗,HRP标记的羊抗猪为二抗,对转印到PDVF膜上的CWP蛋白进行检测,并将重复胶上相应的点进行MALDI-TOF-MS检测。共有有11蛋白点个被检出有免疫原性,用质谱鉴定属于7种蛋白,其中4种为首次报道。自然界中存在的SS2包括有强毒株和无毒株。本试验采用比较蛋白组学的方法,比较了SS2两株强毒株(HA9801和ZY05719)与无毒株T15蛋白二维表达图谱,以期确认和发现SS2毒力标志物。在对强毒株(HA9801和ZY05719)与弱毒株(T15)之间的分泌蛋白的二维电泳图谱比较中,一共发现了九个差异点,其中的八个被生物质谱成功鉴定,其中包括包括MRP、EF、SLY。根据差异蛋白的基因合成引物,以三株SS2菌株DNA为模板进行扩增,其中有5对引物在三株细菌中均扩增出相应基因,一对引物均未扩出,二对仅在强毒株中扩增出。同样采用比较蛋白组学的方法,比较HA9801和ZY05719与T15菌体蛋白的二维表达图谱。在强毒株与弱毒株之间,一共鉴定出3个差异点。根据差异蛋白的基因合成引物,以三株SS2菌株DNA为模板进行扩增,PCR结果表明,此三种蛋白的基因仅在强毒株中分布。用前述的免疫蛋白组学方法,筛选到一种既存在于分泌蛋白中,也存在于膜相关蛋白中的免疫原性蛋白(HM3),用PCR扩增该蛋白的一段基因,定向克隆到表达载体pET-32a(+)中并转化入BL21(DE3)宿主菌。重组菌经IPTG诱导后的SDS-PAGE图谱在46kDa处出现融合蛋白的条带。Western-blotting表明,融合蛋白可被SPF微型猪抗SS2血清所识别,提示该融合蛋白可作为该菌的亚单位疫苗的候选物。更多还原

【Abstract】 Streptococcus suis is a major swine pathogen with a worldwide distribution.Among the 35 serotypes described,SS2 is the serotype most frequently associated with a range of swine diseases including meningitis,pneumonia,septicemia,and arthritis.S. suis is also a zoonotic agent for people in contact with swine or their by-products, causing meningitis,permanent hearing loss,endocarditis,and septic shock[2,3]. From June to August of 2005,an outbreak of a severe invasive infection in humans and pigs occurred in Sichuan Province,China that resulted in 204 human infections and 38 deaths.This incidence followed a much smaller outbreak in 1998 in Jiangsu Province,which killed 14 of 25 patientsLacking suitable vaccine was one of the bottlenecks to control this infection.We describe our work using immunoproteomic approach,a method combining the specificity of antibody with precision of mass spectral analysis,to screen new antigenic proteins from extracellular proteins,membrane-associated proteins and cell-well proteins in SS2.The convalescent serum of a specific pathogen free(SPF) mini-pig recognized nine protein spots on PVDF membranes,which contained extracellurlar proteins of HA9801 and ZY05719 respectively.Antigenic proteins on a duplicate gel of ZY05719 and HA9801 were excised and identified by MALDI-TOF-MS.Peptide mass fingerprinting of the protein spots was performed using the MASCOT server.Sever novel antigenic proteins,as well as some knew antigenic proteins,MRP,EF and SLY, were identified.The membrane-associated proteins(MAP) were enriched using TritonⅩ-114 extraction protocol.These proteins were analyzed with two-dimensional gel electrophoresis(2-DE) and subsequent immunoblotting using hyperimmune serum of SS2-HA9801 immunized SPF mini-pigs.Thirteen antigenic proteins were recognized in the gels of MAP,and corresponding spots on each duplicate gel were excised and analyzed with MALDI-TOF-MS.cell-wall proteins of Streptococcus suis type 2 were extracted by mutanolysin protocol, then analyzed with two-dimensional gel electrophoresis(2-DE) and subsequent immunoblotting using hyperimmune serum of SS2-HA9801 immunized SPF mini-pigs.11 antigenic proteins were recognized in the gels of CWP,and corresponding spots on each duplicate gel were excised and analyzed with MALDI-TOF-MS.Seven immunogenic proteins were identified as significant by Mascot and 4 proteins were reported for the first time in SS2。Lacking suitable virulent markers was another bottleneck to control this infection. Comparative proteomic analysis was chosen to evaluate the differences of 2D gel profiles between two virulent strains(ZY05719 and HA9801) and an avirulent strain (T15).Eight of Nine differential proteins,which including MRP and EF,were successfully identified by MALDI-TOF-MS from the extracelluller proteins of SS2.Primers were synthesized against to the genes of identified proteins,PCR amplification and sequence revealed that genes for seven of the antigenic proteins,with five also in the avirulent strain,were found in both virulent strains and genes for six of differential proteins,with four also in the avirulent stain,were found in both virulent strains.Three differential proteins were also found in the bacterial proteins from the three strains of SS2.Primers were synthesized against to the genes of identified proteins,in order to find difference between virulent strains and avirulent strain at gene level.A immunogenic protein,named HM3,which was identified by using immunoproteomic assay.Partial gene of this protein were amplified form the genome of HA9801 by PCR and inserted into expression plasmid pET-32a(+) after double digested by BamHⅠand SalⅠ,then transformed into BL21(DE3) where they were induced to express by IPTG.After induced,there was specific proteins band of approximately 46kDa on the SDS-PAGE gel.Western-blotting showed that recombinant protein could react with immunized serum of HA9801 of SPF-minipig. This protein could take as a vaccine candidate of SS2。更多还原