【作者】 段志涛；

【导师】 何孔旺； 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 猪链球菌2型（Streptococcus suis serotype 2，SS₂）是我国猪链球菌病的重要病原，不仅可引起猪脑膜炎、关节炎、心内膜炎、败血症、肺炎和突然死亡，还可感染相关人员并致死，是一种重要的人畜共患病病原。 SS2可区分为强毒株、弱毒株和无毒株，已发现的毒力因子主要包括溶菌酶释放蛋白（MRP）、胞外蛋白因子（EF）、溶血素（SLY）、New ORF2、纤维蛋白原结合蛋白（FBP）等。本实验基于GenBank SS2上述5个主要毒力因子的报道序列，分别在其上下游设计引物，应用不同引物对进行PCR扩增测序，对毒力因子进行基因定位，发现上述基因均位于20kb的片段之中，在这些片段中，发现新的可能毒力因子次黄嘌呤核苷酸脱氢酶（IMPDH）。该基因广泛存在于猪链球菌35个血清型当中，其中大部分为致病型，利用PCR技术将这一毒力因子扩增出目的片段构建了含目的基因的重组表达质粒pET-IMPDH，该质粒转化入宿主菌BL21（DE3），体外表达蛋白经镍柱亲合层析纯化后活性染色，证实了该蛋白的生物学活性。 1 SS2 IMPDH的发现及生物学特性鉴定 根据SS2的部分测序结果，发现位于已知毒力基因orf2与mrp之间存在两个新的开放阅读框序列。选取可能含有抗原决定簇肽段对应的核酸序列，该阅读框（2738-3694）编码319个氨基酸残基，分子量为33.5kD，与已知任何基因同源性较低。通过InterPro、PHD、DNAstar分析阅读框，并定向克隆至pET-32α（+）载体中，转化至大肠杆菌BL21，表达出分子量为48kD的融合蛋白。该蛋白免疫转印可被SS2的抗血清识别，具有免疫原性；并且含有IMP dehydrogenase结构域，催化IMP生成XMP；纯化蛋白作用于HEp-2细胞，流式细胞仪检测该蛋白可明显延长细胞的S期。 2 SS2 IMPDH重组蛋白的纯化和抗血清的制备 SS2 IMPDH的重组蛋白含有6个连续的组氨酸残基，利用能与Ni²⁺结合的特性，进行纯化。IMPDH重组蛋白经Ni-NTA His·Bind Resin进行非变性条件更多还原

【Abstract】 Streptococcus suis serotype 2 is the important pathogen of diseases in pigs and is a major cause of meningitis, septicemia, arthritis, and bronchopneumonia in young pigs and can cause meningitis in humans.With Streptococcus suis serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. The virulent factors that can be found include muraminidase-released protein(MRP), extracellular protein factor(EF), suilysin(SLY), New ORF2, fibronection(Fn). Give the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, S. suis serotype 2 was sequenced partly in an effort to identify important virulence factors according to five main virulent factors: MRP, EF, SLY, New ORF2 and FBP in GenBank. The gene encoding IMPDH was found. The PCR product of the open reading frame was transformed into E.coli BL21 and the fusion protein of 48 kD was expressed. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic. IMPDH activity straining confirmed that the protein had IMPDH function and can catalyze the rate-limiting reaction of GTP biosynthesis, the NAD-dependent reduction of IMP into XMP. Flow cytometry(FCM) revealed that the protein had apparent effect on HEp-2 cell cycle. 1 Cloning and Expression of Virulence-Associated Determinants of Streptococcus suis Serotype 2Analysis Gene sequence revealed that a new open reading frame that encoded a polypeptide of 319 amino acid residues with a calculated molecular mass of 33.5 kD was located between orf 2 and mrp of S.suis serotype 2. Second structure was analyzed by InterPro PHD DNAstar software, the PCR product of the open reading frame (2738 — 3694) was transformed into E.coli BL21 and the fusion protein of 48,000MW was expressed. At the amino acid level the deduced primary sequence更多还原