【作者】 谭臣；

【导师】 陈焕春； 贝为成；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2010， 博士

【摘要】 猪链球菌病由猪链球菌（Strreptococcus suis）引起的一种重要细菌性传染病。猪链球菌根据荚膜抗原成分的不同,分为33个血清型。其中猪链球菌2型（Strreptococcus suis serotype 2, SS2）作为一种重要人畜共患传染病,’不仅影响着养猪业健康发展,还严重危害着人类的健康。1998年夏季江苏省暴发了人感染2型猪链球菌病,14人死亡。2005年夏季,在四川省资阳市,暴发了人感染猪链球菌病,204人发病,38人死亡。随着猪链球菌2型对养殖业和人类健康危害的日益加剧,最近几年其已成为国内外研究的热点,尤其是关于其致病与免疫机理方面的研究。致病机理方面的研究主要是集中在毒力因子发掘与功能鉴定以及引起脑膜炎的机制。虽然取得了一定的进展,但是仍然有许多问题没有阐明清楚,尤其是毒力因子之间的相互作用调控网络与细菌致病机理方面的相关性等问题。细菌的双组分调控系统（Two-Component Signal Transduction System, TCSTS）在细菌感受外界环境变化,调节自身基因表达方面发挥着重要作用。随着我国猪链球2型05ZYH33和98HAH12菌株全基因组测序的完成,目前已经有15对双组分调控系统被成功鉴定。本实验室构建了其中一对1660hk/rr新型双组分调控系统基因缺失突变株,通过体内外试验证实该双组分调控系统与猪链球菌2型致病性相关。进一步对一些已报道的重要毒力因子进行了荧光定量PCR验证,证实了研究结果的可靠性。然而,关于1660hk/rr双组分调控系统基因缺失后引起细菌致病性下降的分子机制尚不清楚。鉴于以上背景,本课题在前期构建的1660hk/rr双组分调控系统基因缺失突变株基础上,通过基因表达谱芯片,研究了1660hk/rr基因缺失突变株的基因表达情况,结果显示除缺失目的基因外共有249个基因表达发生了变化。其中SSU05₀153基因下调倍数最大,通过BLAST发现SSU05₀153基因编码内皮素转化酶1（ecel）,与真核生物的内皮素转换酶具有较高的同源性。ecel基因对调节生物体内皮素（ET）的生物学活性起着非常重要的作用。大量研究证实内皮素系统代谢功能紊乱与高血压、脂质代谢紊乱、糖尿病及动脉粥样硬化等大量疾病有关。通过构建猪链球菌2型ecel基因缺失突变株,经过体外生物学特性和动物体内毒力试验,研究ecel基因与毒力的关系。当前,猪用商品化链球菌疫苗主要是灭活疫苗和弱毒疫苗。灭活疫苗副反应大,另外对其它血清型交叉免疫保护率不高。而使用的C群链球菌弱毒疫苗自然致弱,存在毒力返强的风险。因此,寻找新的疫苗抗原,研制新型安全、高效猪链球菌2型基因工程疫苗显得尤为重要。通过生物信息学分析和同源性比对,发现6-磷酸葡萄糖脱氢酶（6PGD）基因在多种细菌中非常保守,且文献已经报道肺炎链球菌的6PGD对小鼠能提供40%的免疫保护。因此,本课题以猪链球菌2型SC19菌株为材料,克隆表达了SS2的6-磷酸葡萄糖脱氢酶,开展了其体外活性和免疫原性研究。论文主要研究内容包括：1.双组分调控系统1660hk/rr基因缺失突变株与野毒株的差异表达基因分析根据Geenbank公布的SS2菌株05ZYH33全基因组序列,定制了SS2全基因组表达谱芯片,比较双组分调控系统1660hk/rr基因缺失突变株与野生菌株之间的差异表达基因。结果显示,有89个基因表达下调了2倍以上,其中25个基因下调表达5倍以上；上调2倍以上41个基因中有4个上调了5倍以上。为了获得更多信息,我们进一步统计了差异表达在1.5倍以上的基因。结果有152个基因下调,99个基因上调,占总基因数的11.4%。通过基因变化倍数以及生物信息学分析,我们推测05SSU0153基因（ecel）可能与猪链球菌2型致病性相关。2.ecel基因缺失突变株的构建根据Genbank中公布的SS2的ecel基因即05SSU0153的序列,以SC19菌株DNA为模板,分别克隆ecel基因上游1034bp和下游1041bp并连接到pMD18T载体,构建克隆载体pMD18T-PS和pMD18T-PX。然后逐步用HindⅢ与SalⅠ、SalⅠ与EcoR I酶切pMD18T-PS和pMD18T-PX,同时逐步用同样酶切的自杀性载体pSET4s,两次连接之后得到自杀性质粒pSET4s-0153。将构建好的自杀性重组转移质粒pSET4s-0153电转化到SS2野生菌株SC19中,通过抗生素和温度双重特征筛选,获得05SSU0153基因缺失菌株,命名Δ0153。通过PCR和测序进一步对基因缺失突变菌株进行了鉴定,结果表明突变株构建正确。3.突变株生物学特性的研究将SS2的ecel基因缺失突变株Δ0153在体外连续传代培养,通过PCR鉴定,证明该缺失菌株能够稳定遗传。在液体培养基中培养A0153基因缺失菌株与SS2野生菌株SC19,每小时取样测定菌液吸光值,根据结果绘制生长曲线,结果表明,A0153基因缺失突变株的增殖能力没有发生明显变化,说明ecel基因的缺失对SS2的体外增殖能力无影响。4.突变株对仔猪的致病性将SS2野生菌株SC19与突变株A0153以5×105cfu/ml,1×106cfu/ml和5×106cfu/ml的剂量,通过耳静脉（1m1）分别感染断奶仔猪。感染后每天观察记录仔猪临床症状,记录死亡情况,并通过病理解剖以及组织切片来评价不同菌株致病性的差异。结果显示突变株A0153攻毒后与野生菌株SC19相比,各不同剂量攻毒组临床症状较轻,死亡率显著降低。表明基因缺失突变株Δ0153对仔猪的致病力明显低于野生菌株SC19。5.6PGD蛋白的克隆表达及细菌黏附抑制试验根据Genbank中公布的SS2的6pgd基因序列,设计引物扩增该基因并连接到原核表达载体pET-28a上。将其转化到大肠杆菌表达菌株BL21（DE3）,经IPTG诱导,得到重组蛋白6PGD,用组氨酸纯化试剂柱纯化。通过Western blot鉴定证实其具有反应原性。重组的6PGD蛋白与Hep2和HeLa细胞相互作用,阳性对照组不加蛋白作用,2小时后,所有试验组加入SS2野生菌株与细胞相互作用。结果显示加入蛋白后SS2对Hep2和HeLa细胞的黏附分别减少了72%和66%。表明6PGD能抑制SC19与细胞的黏附。6.6PGD蛋白对小鼠的免疫保护试验将20只6周龄Balb/C雌鼠,随机分成2组,每组10只。第1组免疫100μL完全弗氏佐剂乳化20μg6PGD蛋白,第2组免疫100μL完全弗氏佐剂为对照组,首免后14d,28d分别进行后二免和三免,剂量和方式同首免。但改为用不完全弗氏佐剂代替完全弗氏佐剂乳化。三免后10d所有小鼠用2.5×108 CFU （0.2mL）的SC19通过腹腔攻毒。一免前、二免前、三免前以及攻毒前,对小鼠尾静脉负压采血,检测血清抗体效价。攻毒后每天观察并记录小鼠的临床表现和死亡情况。结果显示：重组6PGD蛋白免疫小鼠14天可检测到特异性的抗体,在38天时抗体效价达到较高水平。攻毒后接种佐剂对照组小鼠表现出明显的临床症状,48h内全部死亡,解剖后发现多脏器有出血,从各脏器均能分离到攻毒用菌株。而6PGD蛋白免疫组攻毒后最终80%的免疫小鼠得到保护。攻毒后部分小鼠表现出临床症状。存活小鼠解剖后无明显病理变化,从脏器中不能分离到攻毒用野毒菌株。7.6PGD蛋白对仔猪的免疫保护试验将20头4周龄仔猪随机分成2个试验组,第1组每头肌肉注射500μL完全弗式佐剂乳化500μg6PGD蛋白,第2组每头注射500μL完全弗氏佐剂为对照组,两周后进行二免,二免的剂量和方式与同一免相同。用不完全弗氏佐剂代替完全弗氏佐剂。二免两周后用1.0×106 CFU（1mL）的SC19通过静脉攻毒。一免前、二免前和攻毒前对每头猪前腔静脉采血,用ELISA检测血清抗体效价。攻毒后的7天内每天观察临床症状。之后处死存活猪,观察病理变化,取组织作病理切片。结果显示重组蛋白免疫组猪14天可检测到特异性的抗体,在28天时抗体效价达到较高水平。攻毒后接种佐剂对照组猪在攻毒后表现明显的临床症状,48h内全部死亡解剖后发现多脏器有充血、出血,从各脏器均能分离到攻毒用菌株。而6PGD蛋白免疫组攻毒后大部分表现临床症状,部分症状较重,48h内死亡20%,部分猪逐渐好转,最终有50%的猪存活。存活猪,大部分脏器中不能分离到攻毒用野毒菌株。更多还原

【Abstract】 Streptococcus suis is an important swine pathogen and 33 S. suis serotypes have been identified based on differences in their capsular antigens. S. suis serotype 2 (S. suis 2 or SS2) is the most frequently isolated serotype worldwide and can cause meningitis, septicemia, arthritis, endocarditis, pneumonia and even acute death in swine. In addition to causing disease in pigs, it is also an important zoonotic agent for humans in contact with diseased pigs or their products. In 1998, SS2 outbreaks in humans in Jiangsu, China infected 25 people of which 14 died. Between July and August 2005, an outbreak of SS2 infection occurred in Sichuan, China, involving 204 cases, of whom 38 died.Due to high economic losses as well as threat to human life, the study of SS2 pathogenesis and the development of new vaccines is crucial for prevention and control of this disease. In this study, a micorarray was used to detect the differences in gene expression of a SS2 two component signal transduction (TCTS) 1660hk/rr gene deletion mutant and the wild type organism. Results show that totally 249 genes had differential expression patterns. The Endothelin-converting enzyme 1 gene (ecel) was found to be the most downregulated. The ecel gene deletion mutan was constructed by a homologous recombination based method. In vitro and in vivo data demonstrates that the ecel deletion mutan was attenuated. In order to find an effective subunit vaccine the 6-phosphogluconate-dehydrogenase (6pgd) was cloned and expressed. Sequencing this gene found that it has high similarity at the nucleotide level with other bacterial, for example the S. pneumoniae. The recombinant 6PGD protein inhibits the SS2 adhesion to host cells. Immunising animals with the recombinant 6PGD protein provided 80% and 50% protection of mice and swines respectively against SS2 high virulence strain infection. The main research contents include:1. Gene different expression of 1660hk/rr deleted mutant strainIn this study we used a microarry which was made by using available the SS2 05ZYH33 strain whole genome sequence. To compare with the wild type SS2 strain SC19 gene expression, the TCTS 1660hk/rr gene deletion mutant shows that 89 genes were down-regulated 2-fold and 41 genes were upregulated 2-fold. Of the 89 genes,25 were downregulated more than 5 fold and 4 were upregulated to the same level. In order to obtain more information, we altered the cut off gene expression change level to 1.5 times. In total 152 genes were down regulated and 99 were upregulated accounting for 11.4% of all the ORFs within the SS2 genome.2. Construction of ecel mutantPrimers were designed to amplify 1034bp of the ecel gene upstream and 1041bp of the downstream sequence based on the SS2 strain 05SSU0153 ecel gene sequence. The two PCR products were purified and cloned into a pMD18T vector resulting in the construction of pMD18T-PS and pMD18T-PX. pMD18T-PS was digested with HindⅢand Sal I, pMD18T-PX digested with Sal I and EcoR I. The suicide vector pSET4s was digested with the same enzymes gradually, after the ligation was completea suicide plasmid, pSET4s-0153 was obtained. The resulting suicide recombinant plasmid pSET4s-0153 was transformed by electroporation into the SS2 wild strain SC19. Through a double feature selection of antibiotics and temperature, the 05SSU0153 gene deletion mutant strain was obtained and namedΔ0153. The gene deletion mutant was confirmed by PCR3. Characterization of the SS2 mutantsHeredity and stability of the SS2 ecel gene deleted mutantΔ0153 was confirmed by continuous passage culture and PCR this confirmed that the mutation was stable. The growth rate of the ece1 deletion mutant was compared to the wild type strain SC19 by measuring OD600 at different time points. The results indicated no difference between parent strain SC19 and mutant strainΔ0153.4. Virulence in pigsIn order to test the virulence capacity of mutant strainΔ0153, pigs were intravenously infected with three different doses 5×105cfu/ml, 1×106cfu/ml and 5×106cfu/ml of wildtype (SC19) and mutant strainΔ0153. Clinical symptoms and survival were observed and recorded daily after the initial infection. The pathological anatomy and pathology were also used to evaluate the virulence of different strains of SS2. The results showed that compared with wildtype strain SC19, mutant strainΔ0153 post-infection showed mild clinical symptoms of disease and the death rate decreased significantly. All these results suggested the mutant strain A0153 significantly attenuated when compared to the wildtype strain SC19.5. Expression 6PGD protein and the bacterial inhibition testThe 6pgd gene of SS2 was amplified by PCR withprimers designed according to the published Genbank SS2 6pgd gene sequence and cloned to the expression vector pET-28. The plasmid was transformed into E. coli expression strain BL21 (DE3) and induced by IPTG. Recombinant 6PGD protein was isolated using histidine purification reagents and confirmed by western blot. The ability of the 6PGD protein to inhibit SS2 virulence by monitoring adhesion to host Hep2 and HeLa cells was tested. The results showed that after adding the 6PGD protein, SS2 adhesion to Hep2 and HeLa cells was decreased by about 72% and 66%. This may indicate that 6PGD protein may play as an adhesion factor in SS2. 6. 6PGD protein protection in miceTwenty 6-week-old Balb/c female mice were randomly divided into two experimental groups of ten to evaluate the efficacy of the 6PGD protein as a subunit vaccine. Group one was immunized subcutaneously with 20 mg of purified r6PGD mixed with 100 mL complete Freund’s Adjuvant on day 0 (primary immunization). At days 14 and 28, the mice were immunized with 20 mg of r6PGD protein emulsified with 100 mL incomplete Freund’s Adjuvant according to the manufacturer’s protocol. The other group was immunized subcutaneously with adjuvant as a control. Ten days after the third vaccination, the animals from both groups were challenged intraperitoneally with 2.5×108 CFU per mouse of log-phase SS2 virulence strain SC19 in 0.2 mL TSB. Mice were bled at days 0,14,28 and 38, serum samples were detected by 6PGD ELISA. Clinical symptoms and survival were monitored daily after infection. The results showed that anti-6PGDantibody can be detected at day 14 and that on day 38 the specific antibody reaches a higher level. After infection, the adjuvant control mice showed significant clinical symptoms and all died in 48h. Anatomicallymultiple organ bleeding was noted and SS2 could beisolated from various organs. Some of the 6PGD protein immunized group mice showed clinical symptoms of disease but finally 80% were protected. Afternatomical examination of the surviving mice, no significant changes could be observed and the challenge organismcould not be isolated from organs.7. 6PGD protein protection in pigsTwenty 4-week-old pigs were taken from a herd that was SS2 free and were randomly assigned to two groups of 10 pigs each. Group one were immunized intramuscularly with 500 mg of purified 6PGD mixed with 500 mL complete Freund’s adjuvant on day 0. At day 14, the pigs were immunized with 500 mg 6PGD emulsified with 500 mL incomplete Freund’s adjuvant, according to the manufacturer’s protocol. The other group was immunized with only the adjuvant as the control group. Two weeks after the booster immunization, all twenty pigs in both immunizedcontrolgroups were intravenously challenged with 1×106 CFU per pig of a log-phase culture of SS2 virulence strain SC19 in 1 mL TSB. On days 0,14, and 28, blood samples were obtained from each pig by precaval vein bleeding and serum samples were detected by 6PGD ELISA. Clinical symptoms and survival were monitored daily after infection. After 7 days observation, all the surviving pigs were killed to observe the pathological changes in the organs and also take the tissues for pathology studies. The results showed that the anti-6PGD protein specific antibody could be detected on day 14 and that on day 28 the antibody reaches a higher level. After infection, the adjuvant control pigs showed significant clinical symptoms and all died at 48h. Anatomically the animals were foundto have multiple organs with hyperemia, hemorrhage and the challenge organism could be isolated from various organs. In the 6PGD protein immunized group, some pigs had significant clinical symptoms and 20% died within 48h. Finally,50% of the immunized pigs were protected. After anatomical examination of the surviving pigs, most of the organs did not contain culturable SS2.更多还原