【作者】 吴全忠；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2001， 博士

【摘要】 用豚鼠建立了猪链球菌2型（Streptococcus suis Type 2）引致脑炎和败血症的动物模型，研究了猪链球菌2型在动物模型体内的动态分布。并将该菌毒力相关的溶菌酶释放蛋白基因（mrp）克隆到真核表达载体构建了真核表达质粒，并研究其对豚鼠的免疫效果。 1 选用对猪具有致病性的猪链球菌2型江苏分离株HA9801感染豚鼠，引致脑炎及败血症，建立了动物模型。结果显示16～24日龄的豚鼠较易感，最佳感染途径是皮下注射，鼻腔、腹腔次之。感染豚鼠表现出典型脑炎的临床症状及病理变化。经重复试验检测了该菌株对豚鼠的LD₅₀，从感染死亡豚鼠体内包括脑脊液、大脑、血液及各实质器官均可分离出该病原菌，与仔猪自然感染情况相似。该动物模型的建立，为进一步探究猪链球菌2型感染的体内动态分布、感染机制、毒力因子及免疫应答奠定了基础。 2 用猪链球菌2型江苏分离株HA9801，皮下或鼻腔接种豚鼠，检测体内动态分布。结果显示：皮下接种后，该菌最早可于28小时到达心血，32小时还可在肺、脾及扁桃体中分离到，32～36小时除上述器官外还见于肝、肾、脑、脑脊液，并引发神经症状而致豚鼠死亡，接种后120小时心血和扁桃体阳性率分别可达61.5％和69.2％。鼻腔接种同居感染表明，该菌很快传播给其它豚鼠，第8天鼻腔拭子阳性率100％（10/10）；感染豚鼠心血细菌检查结果表明，第9天50％呈阳性，第12天70％（7/10）呈阳性。脑组织检查结果显示，只有出现神经症状及死亡的豚鼠，才能从脑脊液和脑组织中分离到细菌，说明细菌突破血脑屏障，是引发脑炎的前提。 3 将含有猪链球菌2型mrp基因的质粒pMR11，在大肠杆菌中表达了136KD的该菌溶菌酶释放蛋白（MRP，muramidase-released protein），将表达菌用超声波破碎制成的抗原，免疫豚鼠，通过免疫转印检测到MRP的特异抗体，用PPA-ELISA对检测了血清效价；攻毒表明表达蛋白对猪链球菌2型有一定的保护作用。 南京农业大学 博士学位论文 4用地高辛分别对猪链球菌2型的溶菌酶释放蛋白基因（mrp）和胞外蛋白基因（epf）进行了高效标记，结果探针特异敏感，提取23株猪源涟球菌和1株人源的猪链球菌共24株菌的MA，进行斑点杂交检测，结果mrp／mrp＊和ePf／ePf＊阳性率均为 58．33％（14／24），PCR结果和 ELISA检测 MRP结果一致。表明地高辛标记的核酸探针，可用于猪链球菌2型的分子流行病学涉调查。 5将猪链球菌2 型编码MRP的mrn基因，插入到真核表达载体pcDNA3．1(一MccHisC质粒CMV启动于的下游，构建了真核表达质粒pCMR。应用PCR技术能扩增出885hp的mrp基因的特异条带，通过双酶切鉴定克隆方向正确，将该质粒用脂质体转染方法转染卯／0细胞，应用RT－PCR、免疫转印和免疫细胞组化染色，鉴定其得到了表达，为进一步在体内研究mrp基因的功能和作用机制奠定了基础。 6用脂质体包裹的含有猪链球菌 2型 mrp基因的真核表达质粒（pcMR）及该菌 HA980株全菌灭活抗原，分别免疫模型动物豚鼠，肌肉注射，通过厂PA－ELISA检测抗体的产生。免疫后，豚鼠产生MRP抗体滴度呈上升趋势；免疫 2用后，MTT法检测脾淋巴细胞转化率（SI）、腹腔巨噬细胞吞噬活性（AMA），基因免疫引起的细胞兔疫比全菌兔疫组强烈。兔疫后第 16天，各组豚鼠接种 HA9801攻毒，结果显示，mro基因免疫效果虽不如全菌疫苗组，但对豚鼠有一定的保护力，说明MRP具有一定的保护性功能更多还原

【Abstract】 An animal model of Streptococcus suis type 2 causing meningitis and septicaemia was established with guinea-pigs. The dynamic distribution of the bactera in guinea pigs model was investigated. The mm gene coding muraminidase-released protein (MRP) of Streptococcus suis type 2 was inserted into the plasmid pcDNA3.1(-)MycHisC to construct eukaryotic expression plasmid. The immune effect of the DNA vaccine to guinea- pigs and the function of MRP were evaluated. 1 Guinea-pig was established as an animal model, which was induced meningitis and septicaemia experimentally with isolate HA980 1 of Streptoco- ccus suEs type 2. The results showed that young guinea-pigs before weaning were susceptible. The optimum route of inoculation was determined to be hyodermic inoculation. Clinical signs and pathological changes of an acute meningitis were observed. LD50 of HA9801 to guinea-pigs had been determined. The organisms could be reisolated from the brain, cerebrospinal fluid and other organs. All of the above was same as that of piglets infected naturally. Guinea-pig model could be useful for further research on Streptococcus suis type 2 infection. 3 E~k*~ M眫i~ 2 The dynamic distribution of Jiangsu isolate HA9801 of Streptococcus suEs type 2 in guinea pigs model was investigated by the hyodermic and intranasal infections. The results showed the organisms could be reisolated from heart blood at 28 hours after hyodermic inoculation. The bacteria could be reisolated from lung, spleen and tonsil at 32 hours afler inoculation, too. Between 32?6 hours, the organisms could transmit to liver, kidney, cerebrospinal fluid and the brain. The guinea pigs suffered obvious nervous signs and were rapidly to death. It showed the reisolation positive rates of heart blood and tonsil were 61.5% and 69.2% at 120 hours. After intranasal inoculation, 50% of guinea pigs were found to have positive nasal swab at the 4th day after inoculation, 100% at the 8th day. Heart blood samples of guinea pigs were 50% positive at the 9th day, and 700/o positive at the l2th條8th day. The organisms could be reisolated from the cerebrospinal fluids and brains of the guinea-pigs which showed clinical nervous signs and death. 3 The plasmid pMRI 1 harbouring mrp gene of Streptococcus suis type 2 was transformed into E.coli and expressed protein of 136 KD. The expressed protein was used as antigen by broken E. co/i with ultrasonic disintegrator and immunized animal model guinea pigs. Immunoblotting and PPA-ELISA were used to detect specific anti-MRP antibody and the titers of anti-serum, respectively. The expressed protein could protect partly guinea pigs against Streptococcus suis type 2 infection. 4 Muraminidase-released protein gene (mrp) and extraclluar protein factor gene (epf) of Streptococcus stiEs type 2 were labeled with DIG-dUTP, respectively. Both of the probes had specificity and sensitivity. DNAs of 24 Streptococcal strains were detected by dot-blot using these probes. The results showed that the positive rates of mrp/mrp* and epf/epf?were same of 58.33% (14/24) . It showed that dot-blotting with DNA probes labled by DIG-dUTP could be used to investigate the molecular epidemiology of Streptococcus suEs type 2. 5 The mrp gene coding MRP of Streptococcus suis type 2 was inserted into the plasmid pcDNA3.1(-)MycHisC, under the cytom更多还原