摘要
猪链球菌2型(Streptococcus suis serotype 2; SS2)感染已经成为一种严重的人畜共患病,不仅造成大量猪死亡,更严重威胁着人类健康。我国于1998年和2005年两次在江苏和四川暴发人感染猪链球菌2型疫情,分别造成14人和39人死亡。不同于国外人感染病例以脑膜炎为主的特点,我国死亡病例多为休克型,病例起病急,病情重,病程短,死亡率高。
目前对我国SS2的致病机制知之甚少,目前已知的毒力因子还不能全面解释毒素休克综合征(TSS)的发生,而对致病机制的缺乏了解又阻碍了SS2疫苗的研究。已知的毒力因子和保护性抗原包括荚膜多糖(CPS)、溶菌酶释放蛋白(MRP)、胞外因子(EF)、溶血素(SLY)、烯醇酶、6-磷酸葡萄糖酸脱氢酶(6PGD)、Sao等。在此基础上发现更多、更有效的保护性抗原成为SS2疫苗研究工作的当务之急。
本研究通过免疫蛋白质组学识别出SS2菌株98012细胞壁和上清蛋白中的32种免疫原性蛋白,其中8种为细胞壁蛋白、5种为非细胞质蛋白(Non-Cytoplasm Protein)、其它为胞质蛋白。使用比较蛋白质组学方法识别出我国高致病株与普通致病株差异表达蛋白,并进行后续验证。选择其中的10种蛋白以pET-32a(+)为载体重组表达,表达已知保护性抗原MRP为后续研究阳性对照。免疫大鼠获得抗血清,Western Blot验证10种蛋白在16种不同SS2菌株中的存在状况,发现SSU98_0171、SSU98_1094、SSU98_1549、SSU98_1819、SSU1664五种抗原在不同菌株间高度保守,SSU98_0156、SSU98_0197、SSU98_0201、SSU98_0267、SSU98_1675在不同菌株之间表达有差异。分析比较蛋白质组学结果以及后面的验证结果,并与不同菌株的致病力相结合,笔者将SSU98_0156、SSU98_0197、SSU98_0267、SSU98_1675这4种蛋白定义为不同致病力菌株之间的差异表达蛋白,其中SSU98_1675为国内高致病株与国外致病株之间的差异蛋白,SSU98_0197、SSU98_0267在不同菌株间存在多态性。
随后对除SSU98_0156之外的其它10种重组抗原进行评价。比较6名病人和5名健康人血清中针对10种重组抗原的抗体差异,结果显示SSU98_0197、SSU98_1094、SSU98_1549、SSU1664、MRP的抗体水平在病人比健康人显著性升高。比较针对10种重组抗原的抗体在人工感染小型猪前后血清中的变化情况,结果显示SSU98_0197、SSU98_1094、SSU98_1549、SSU98_1675、SSU1664的抗体在感染后比感染前显著性升高。比较针对10种重组抗原的抗体在灭活全菌免疫小型猪前后的变化情况,结果显示SSU98_0197、SSU98_0267、SSU98_1094、SSU98_1549、SSU1664、MRP的抗体在免疫后比免疫前显著性升高。体外大鼠抗血清调理吞噬试验显示有6种蛋白的抗血清调理杀菌率在50%以上,分别是SSU98_0171、SSU98_0197、SSU98_0267、SSU98_1094、SSU98_1819、SSU1664。
根据前期试验结果,选择7种抗原以pET-28a(+)为载体重新表达,以去除TRX(硫氧还蛋白)对动物试验的影响,成功表达出5种抗原。使用QS-21(皂角甙的精细提取物)为佐剂,在CD-1小鼠模型上验证5种抗原的保护效果。首先对5种抗原单独免疫保护效果进行评价,使用1.5×LD50的98012菌液攻毒, SSU98_0197、SSU98_1094、SSU98_1675、MRP免疫组有较高存活率,分别为70%、67%、60%、70%,与单纯QS-21免疫组比较,经Wilcoxon检验分析,均有显著性差异,而SSU1664免疫组没有保护性。增加攻毒量后SSU98_0197、SSU98_1094、SSU98_1675、MRP免疫组存活率均下降,分别为20%、20%、50%、20%。与单纯QS-21免疫组比较,均有显著性差异,而SSU1664免疫组仍然没有保护效果。
对保护效果好的SSU98_0197、SSU98_1094、SSU98_1675尝试使用组合抗原免疫小鼠。使用6.6×LD50的98012菌液攻毒,分组为SSU98_0197、SSU98_1094、SSU98_1675、SSU98_0197与SSU98_1675组合、SSU98_1094与SSU98_1675组合、SSU98_0197与SSU98_1094组合、三种蛋白组合免疫,7个试验组,存活率分别是30%、30%、30%、50%、40%、30%、60%,与单纯QS-21免疫组比较,经Wilcoxon检验分析,均有显著性差异。7个免疫组之间比较,仅三种蛋白组合免疫组与SSU98_0197单独免疫组有显著性差异。进一步比较SSU98_0197、SSU98_1094和SSU98_1675三种蛋白组合免疫组以及SSU98_0197和SSU98_1675两种蛋白组合免疫组的保护效果。4.8×LD50的98012菌液攻毒时,三种蛋白组合免疫组,SSU98_0197与SSU98_1675组合免疫组的存活率分别是60%和40%,两组间无显著性差异。使用7×LD50的98012菌液攻毒,三种蛋白组合免疫组和SSU98_0197+SSU98_1675组合免疫组的存活率分别是50%和20%,经Wilcoxon检验分析,两组间有显著性差异,提示三种蛋白组合免疫组对高剂量菌液的攻毒仍然有保护效果。
将筛选出的3种保护性抗原SSU98_0197、SSU98_1094、SSU98_1675命名为SIP1、Lpp36、SIP2。构建基因缺失突变株05ZYΔLpp36和05ZYΔSIP2。为后续的功能研究奠定基础。
Streptococcus suis serotype 2 (SS2) infection has become a public health concern due to its zoonotic capability to cause severe infections in slaughterhouse workers and those who handle infected pork. Two outbreaks have occurred in humans in China with high mortality (19%-56%). One that happended in 1998 invovled 14 humans who died out of 25 infected cases. Another one that happended in 2005 included 215 reported cases and 39 deaths. In these outbreaks, toxic shock syndrome (TSS) was a major etiological and mortality factor.
The mechanisms involved in the pathogenesis and virulence of S. suis are not completely understood, and attempts to control the infection are hampered by the lack of an effective vaccine. The known virulence factors and protective antigens of S. suis includes CPS, muramidase-released protein (MRP), extracellular factor (EF), suilysin (SLY),Enolase, 6-phosphogluconate-dehydrogenase (6PGD), Sao, etc. Some of these proteins had been proved to be able to provide certain degree of protection against the challenge of S. suis but not good enough yet.
In this study, using an immunoproteomic approach, we identified 32 cell wall proteins (CWPs) and extracellular proteins (ECPs) with high immunogenicity in SS2. Among these proteins, eight known cell wall attached proteins, including 6 proteins containing the LPXTG motif, were identified in this work. The other proteins includes 5 non-cytoplasm proteins, and 19 cytoplasm proteins. Some differentially expressed proteins were identified by comparative proteomic analysis between the highly pathogenic SS2 and the moderately pathogenic SS2, and the differences were confirmed in the following study.
Ten selected proteins were expressed in E. coli, and the amplified gene sequences were inserted into pET-32a(+) and transformed into E. coli strain BL21 (DE3). At the same the time, MRP was expressed as the positive control for further study. The polyclonal antisera against each recombinant protein were obtained from Wistar rats, and the immunized rats were bled 7 days after the final immunization. To determine the prevalence of those selected proteins in different SS2 strains, CWPs of 16 different SS2 strains were analyzed by Western blot with specific antiserum against each of the 10 recombinant proteins. Five proteins (SSU98_0171, SSU98_1094, SSU98_1549, SSU98_1819 and SSU1664) were detected in all 16 strains tested. SSU98_0201 were not detected in S. suis 1330. SSU98_0156, SSU98_0197, SSU98_0267, SSU98_1675 were defined as differentially expressed proteins between differently pathogenic SS2. SSU98_1675 could be used to distinguish the domestic highly pathogenic SS2 and foreign pathogenic SS2, while SSU98_0197 and SSU98_0267 size variants were expressed in differently pathogenic SS2.
The recombinant antigens except SSU98_0156 were further evaluated. We tested the reactivity of 6 patient sera as well as 5 healthy individuals to the recombinant antigens. Besides MRP, patients showed significantly higher antibody levels against 4 antigens (SSU98_0197, SSU98_1094, SSU98_1549 and SSU1664). Furthermore, we assessed the specific antibody responses to the recombinant proteins by using sera isolated from both infected piglet and piglets immunized with formaldehyde-killed bacteria. After experimental infections, protein SSU98_0197, SSU98_1094, SSU98_1549, SSU98_1675 and SSU1664 induced significant antibody responses. In comparison to the sera before immunization, specific antibodies against protein SSU98_0197, SSU98_0267, SSU98_1094, SSU98_1549 and SSU1664 were significantly increased in the immunized piglets. Opsonophagocytic killing abilities of specific antibodies against the recombinant antigens were evaluated by the standard bactericidal assay. Antisera against 6 proteins (SSU98_0171, SSU98_0197, SSU98_0267, SSU98_1094, SSU98_1819 and SSU1664) enhanced the inhibition of S. suis growth in human blood.
Taken together, 7 antigens were selected for futher screening protective antigens. Addtionally, to avoid the interference of TRX in the protective capacity assay, we expressed 7 antigens using pET-28a(+) expression vector, and 5 antigens were expressed and purified successfully. CD-1 mice were immunized with recombinant antigens mixed with QS-21. The mice were challenged with 1.5×LD50, respectively, the survival rates of groups immunized with SSU98_0197, SSU98_1094, SSU98_1675, MRP were 70%, 67%, 60%, 70%. When the mice were challenged with higher dose, the survival rates decreased to 20%, 20%, 50%, 20%. In both protection experiments, the statistical differences between recombinant antigens vaccinated groups and QS-21 vaccinated group were significant, while SSU1664 had no protective capacity.
Then SSU98_0197, SSU98_1094, SSU98_1675 were assembled to achieve better protective effects. The mice were challenged with 6.6×LD50, and the survival rates of groups immunized with SSU98_0197, SSU98_1094, SSU98_1675, SSU98_0197 plus SSU98_1675, SSU98_1094 plus SSU98_1675, SSU98_0197 plus SSU98_1094, 3 proteins combined vaccine were 30%、30%、30%、50%、40%、30%、60%, respectively. And the statistical difference between 3 proteins combined vaccinated group and SSU98_0197 vaccinated group was significant.
Furthermore, the protective capacity of 3 proteins combined vaccine and SSU98_0197 plus SSU98_1675 combined vaccine was evaluated. When the mice were challenged with 4.8×LD50, respectively, the survival rates of groups immunized with 3 proteins combined vaccine, SSU98_0197 plus SSU98_1675 combined vaccine were 60% and 40%, respectively. The statistical difference was not significant. When the mice were challenged with 7×LD50, respectively, the survival rates decreased to 50% and 20%, and the statistical difference was significant. This indicated that the 3 proteins combined vaccine had the better protective capacity against high dose challenge.
The protective antigens SSU98_0197, SSU98_1094, SSU98_1675 were named SIP1, Lpp36, SIP2. Two deletion mutants 05ZYΔLpp36 and 05ZYΔSIP2 were constructed successfully.
引文
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