猪链球菌2型侵袭脑微血管内皮细胞相关毒力因子的筛选
毕业论文关键词：猪链球菌2型；毒力因子；侵袭；人脑微血管内皮细胞
Screening of virulence factors associated with invasion of brain micrvascular endothelial cells in Streptococcus Suis Serotype 2
Abstract: Meningitis pathogens crossing the blood-brain barrier process is the important step related with meningitis caused by the bacteria, brain microvascular endothelial cells is the important component of blood-brain barrier, so we used cell invasion experiments to screen related genes of Streptococcus suis through blood-brain barrier initially. In order to explore the related genes of SS2 to invade brain microvascular endothelial cells, the research randomly mutated the genome of ZY05719 strain by pMar4s vector carrying transposon TnYLB-1, and the mutant library containing 100 mutants was successfully constructed. One mutant (P < 0.05) with reduced ability to invade was screened by hMEBC invasion test. The result showed that the invasion rate of the mutant was 22.83 times lower than the wild strain, and then the insertion site of the transposon in the mutant was identified and analyzed by reverse PCR . The silencing gene inserted by transposon was found to be a ABC transporter permease gene (ZY05719_RS06545). This study will provide a foundation to the research of invasion of brain microvascular endothelial cells mechanism by SS2.
Key words: Streptococcus suis serotype 2；virulence factors；invasion；hBMEC
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法2
1.1 材料2
1.1.1 菌株、质粒和细胞2
1.1.2 引物、试剂和仪器2
1.2 猪链球菌2型转座子突变体文库的构建3
1.2.1 pMar4s质粒提取3
1.2.2 猪链球菌感受态的制备3
1.2.3 电转化4
1.2.4 转座突变株的筛选与鉴定4
1.3 猪链球菌2型侵袭能力减弱突变株的筛选4
1.3.1 侵袭能力减弱突变株的定性筛选4
1.3.2 侵袭能力减弱突变株的定量筛选5
1.4 猪链球菌2型侵袭能力相关基因的鉴定5
1.4.1 基因组DNA的提取5
1.4.2 基因组DNA的酶切及酶切产物的回收6
1.4.3 酶切产物的连接6
1.4.4 反向PCR检测6
2 结果与分析7
2.1 猪链球菌2型转座子突变体文库的构建7
2.2 突变株转座子TnYLB-1的检测7
2.3 猪链球菌2型侵袭能力减弱突变株的筛选8
2.4 猪链球菌2型侵袭能力相关基因的鉴定9
3 讨论 9
致谢10
参考文献10
猪链球菌2型侵袭脑微血管内皮细胞相关毒力因子的筛选 猪链球菌（Streptococcus suis，SS）是革兰氏阳性菌，根据荚膜抗原的差异可以分为35个血清型（1-34 及 1/2）[1]。2 型猪链球菌（SS2）被认为是猪链球菌35个血清型中分布最广、致病性最强的一个血清型，可以引起断奶仔猪支气管炎、关节炎、脑膜炎及败血症等[2]。SS2感染人体之后可导致化脓性关节炎、脑膜炎、心内膜炎和中毒性休克综合症[3]等症状。由此可见，SS2 不仅给养猪业造成巨大的经济损失，也给人类健康带来严重的威胁，故引起了国内外学者的广泛关注。目前研究比较清楚的SS2毒力因子包括荚膜多糖(Capsular polysaccharide，CPS)、溶菌酶释放蛋白(Muramidase-released protein，MRP)和细胞外因子(Extracellular factor protein，EF)、溶血素(Suilysin，Sly)、黏附素、IgG结合蛋白、谷氨酸脱氢酶、透明质酸酶以及细菌样抑制性物质[4]。猪链球菌参与侵袭脑微血管内皮细胞的毒力因子较少有被发现，有报道指出猪链球菌具有有限的侵袭能力[5]，有报道指出猪链球菌根本无侵袭能力[6]，也有报道指出猪链球菌仅在无荚膜时具有侵袭能力[7]，因此通过研究筛选与猪链球菌侵袭脑微血管内皮细胞相关的毒力因子，有助于解释猪链球菌引起脑膜炎致病过程和机制。