论文总字数：17653字

摘 要

2型猪链球菌是一种可以感染猪群使猪体患病同时使人类可以通过接触病猪感染的疾病。从二十世纪末至二十一世纪初，我国内就出现了两次死亡率明显高于一般传染病的SS2疫情事件，引起了国内外对于2型猪链球菌的高度重视，也对2型猪链球菌的致病机理开始着重分析。

各位学者对于2型猪链球菌的不同毒力因子的不同毒性也分别做了详细的研究报告，内化素(英文名称是internalins)是被最早分离研究的一类关于细菌吸附在宿主细胞并对宿主细胞中的受体一起破坏其上皮屏障，促使细菌侵入的一种毒力因子，研究证实了这一种毒力因子在单核细胞增生性李斯特菌中特定的发挥了它的重要作用。本实验为了证明所做假设2型猪链球菌中的内化素与LM中的内化素蛋白发生的作用机制相似，所以选用SS2 05ZYH33为实验菌株，实验采取SSU05-1577基因片段作为目的基因，将基因组测序的结果SSU05-1577片段编码产物为推定的内化素A前体蛋白，因为内化素是否在SS2入侵宿主细胞过程中发挥作用，尚未有相关研究。本课题拟对该内化素SsInlA蛋白基因进行克隆，为后续验证其在侵袭细胞中的毒力作用打基础。

我们预测2型猪链球菌的内化素的机制作用，所以对内化素进行生物信息学分析，分析了内化素中鸟嘌呤和胞嘧啶两者碱基的含量，还有对内化素的同源蛋白的搜索和系统进化树的结果进行讨论，是否两种内化素属于同一类。为后期构建互补菌株以及基因缺失菌株，分析内化素A在2型猪链球菌05ZYH33菌株中的作用提供了生物学信息基础。发现肺炎链球菌和SS2序列的相似性从而猜测二者有相同的祖先，分析结果也表明了SsInlA 很可能是位于细胞表面的一个多功能蛋白质，在 SS2 入侵宿主上皮细胞过程中扮演重要角色。。本实验预测2型猪链球菌05ZYH33的内化素A推测出其分子间的结构和功能类似。进一步，我们对于2型猪链球菌05ZYH33 SsInlA基因片段进行克隆分析。实验根据基因序列设计和合成引物通过PCR技术扩增SSU05-1577基因（Ss2InlA)，选择克隆载体，并构建阳性重组载体质粒T-SsInlA。通过克隆获得的重组载体，来分析2型猪链球菌中内化素A的生化生理结构，包括蛋白质的结构以及内化素中所拥有的氨基酸种类，目的是为了最终能够制备到2型猪链球菌的抗体以及研究其在宿主细胞中如何发挥毒力作用的原理完成前期工作。

关键词： 2型猪链球菌 内化素 克隆 PCR

Informatics analysis and cloning of Internalins of Streptococcus suis type 2

ABSTRACT

Streptococcus suis type 2 is a disease that can infect pigs and make pigs sick while allowing humans to be infected by contact with sick pigs. From the end of the 20th century to the beginning of the 21st century, there have been two SS2 outbreaks in China where the mortality rate is significantly higher than that of common infectious diseases. The pathogenic mechanism of streptococcus began to focus on analysis.

Scholars also made detailed research reports on the different toxicities of different virulence factors of Streptococcus suis type 2 The receptor in the host cell together destroys its epithelial barrier, which promotes the invasion of a virulence factor. Studies have confirmed that this virulence factor specifically plays its important role in Listeria monocytogenes. In this experiment, in order to prove the hypothesis, the mechanism of action of internalizing protein in Streptococcus suis type 2 and internalizing protein in LM is similar, so SS2 05ZYH33 was selected as the experimental strain, and the SSU05-1577 gene fragment was used as the target gene The result of genome sequencing SSU05-1577 fragment encoding product is a putative internalizing protein A precursor protein, because whether internalizing agent plays a role in SS2 invasion of host cells, there has not been relevant research. This subject intends to clone the internalizing protein SsInlA protein gene, and lay the foundation for the subsequent verification of its virulence in invasive cells.

However, we predicted the mechanism of the internalization of Streptococcus suis type 2 based on my selection of bioinformatics analysis of internalization， analysis of the content of guanine and cytosine bases in internalization, The search for homologous proteins of chemokines and the results of phylogenetic trees are discussed to see whether the two internalizers belong to the same category. For the later construction of complementary strains and gene-deficient strains, the analysis of the role of internalization A in Streptococcus suis type 2 05ZYH33 strain provides a basis for biological information. The similarity of the sequence between S. pneumoniae and SS2 was found to guess that the two have the same ancestor. The analysis results also indicate that SsInlA is likely to be a multifunctional protein on the cell surface, which plays an important role in the process of SS2 invading host epithelial cells. This experiment predicted that Streptococcus suis type 2 05ZYH33's internalizer A speculated that its molecular structure and function were similar. Further, we cloned and analyzed the SZInHA gene fragment of Streptococcus suis type 2 05ZYH33. The experiment designed and synthesized primers to amplify the SSU05-1577 gene (Ss2InlA) by PCR based on gene sequence, selected cloning vector, and constructed a positive recombinant vector plasmid T-SsInlA. The recombinant vector obtained by cloning was used to analyze the biochemical physiology structure of endogenin A in Streptococcus suis type 2, including the structure of protein and the types of amino acids possessed by endogenin. Cocci's antibodies and the principle of studying how they exert virulence in host cells complete the preliminary work.

KEYWORDS:

Streptococcus suis type 2 Internalins clone PCR

目 录

摘 要 I

ABSTRACT i

目 录 1

第一章 绪 论 1

1.1 猪链球菌概述 1

1.2 猪链球菌毒力因子 1

1.2.1 表面蛋白和分泌蛋白 1

1.2.2 酶类毒力因子 3

1.2.3 其它新型毒力因子 3

1.3 黏附侵袭毒力因子内化素A 3

第二章 Ss2InlA 片段的生物学分析 5

2.1实验方法： 5

2.1.1 猪链球菌内化素A基因 SsInlA 的一般特性分析 5

2.1.2 SsInlA 的同源性检索以及系统进化树对比 5

2.1.3 猪链球菌SsInlA 与单增李斯特菌InlA的比对分析 6

2.2 分析结果 6

2.2.1 SsInlA 基因的一般特性分析结果 6

2.2.2 SsInlA 基因的同源性检索以及系统发生树的绘制 6

2.2.3 猪链球菌SsInlA 与单增李斯特菌InlA的结构比对分析结果 7

第三章 Ss2InlA片段克隆实验 9

3.1 实验试剂及设备 9

3.1.1 实验试剂 9

3.1.2 主要实验仪器与设备 9

3.2 实验方法 10

3.2.1 菌株的活化培养 10

3.2.2 革兰氏染色及镜检 10

3.2.3 引物设计 10

3.2.4 PCR扩增 11

3.2.5 核酸电泳实验 12

3.2.6 切胶回收纯化 12

3.2.7 加A 13

3.2.8 T载体连接及鉴定 14

3.3 实验结果 15

3.3.1 PCR扩增结果 15

3.3.2 切胶回收结果 16

3.3.3 T载体连接PCR鉴定结果 16

第四章 结论与展望 18

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