【作者】 王楷宬；

【导师】 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】 根据已发表的猪链球菌2型（SS2）3-磷酸甘油醛脱氢酶（GAPDH）基因序列，设计合成一对引物，对包括猪链球菌2型的39株链球菌gapdh进行检测，各种链球菌均能检出此基因，得到预期的1040bp的PCR产物，仅无毒株T15未检出。对HA9801，ZY05719株的gapdh测序，登录GenBank。经BLAST比对和DNAStar软件分析，2株菌的gapdh与GenBank中已发表的SS2的gapdh同源性为99.9％、99.8％。gapdh在链球菌中广泛存在，并高度同源。 对猪链球菌2型四川分离株ZY05719的gapdh全基因进行PCR扩增，并将此基因定向克隆至原核表达载体pET-32a（+）中，构建了表达载体pET-32a-GAPDH。利用大肠杆菌BL21成功表达了58kD的GAPDH融合蛋白，并利用Novagen公司的His·Bind^? Purification Kn（70239-3）纯化出GAPDH蛋白。 利用HEp-2细胞对GAPDH的黏附特性进行研究。ZY05719（GAPDH⁺）和T15（GAPDH^-）虽均能黏附于HEp-2细胞，但GAPDH⁺菌株的黏附菌数显著高于GAPDH^-株（P＜0.05）。纯化的原核表达的GAPDH融合蛋白能显著降低GAPDH⁺株的黏附菌数（P＜0.05），但不能阻断。对GAPDH^-株无抑制作用。证明利用原核表达系统表达的GAPDH具有黏附作用。 基于猪链球菌2型（SS2）通过其胞外蛋白酶裂解猪流感病毒血凝素（HA）的假设，以MDCK细胞为载体，利用观察细胞病变、培养上清血凝素效价的测定、免疫荧光技术，证明SS2江苏分离株产生的胞外蛋白酶能增强猪流感病毒H3亚型广东分离株（A／Swine／Guangdong／4／2003）在MDCK细胞上出现的细胞病变及免疫荧光强度，且MDCK细胞感染病毒的上清HA效价有明显升高。更多还原

【Abstract】 A pair of primer was synthesized based on the published Streptococcus suis type 2(SS2) glyceraldehyde-3-phosphate dehydrogenase gene (gapdh) and PCR was applied to detect 39 streptococcal strains including SS2 .The gapdh was found in all tested strains, but not in SS2 T15. The gapdh of HA9801 ZY05719 and SS2-D were sequenced and logged in GenBank. The gapdh of the above three strains were analysised by BLAST and DNAStar with published SS2 gapdh in GenBank. The homology of them was 99.9%, 99.8% and 99.7%. GAPDH gene was ubiquitous in Streptococci.The gapdh of SS2 Sichuan isolation ZY05719 was cloned by PCR and inserted in prepared vector pET-32 a (+).The recombined vector pET-32a-GAPDH was introduced in E. coli BL21.A 58 kD GAPDH protein was expressed and purified by His·Bind~R Purification Kit (70239-3).An adherence assay with SS2 and HEp-2 cells pre-incubated with purified GAPDH and non-incubated cells was showed a significant reduction in the adhesion of SS2 in the purified GAPDH pre-incubated cells compared to the non-incubated cells. The GAPDH protein of SS2 seems to be involved in the bacterial adhesion to host cells.The aim of the study was to certify whether the extracellaluar proteases of Streptococcus suis 2 type (SS2) Jiangsu isolation could enhance the infection ability of H3N2 Swine Influenza virus (SIV) A/Swine/Guangdong/4/2003, based on the hypothesis that extracellaluar proteases produced by SS2 can cleave the hemagglutinin(HA) of SIV. MDCK cells were used for study model. CPE, haemagglutination litre of cell culture supernatants and immunoflourescence intensity of the cells were all enhanced when the extracellaluar proteases of SS2 existed .更多还原