【作者】 高玉敏；

【导师】 呼和巴特尔； 韩润林；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2009， 博士

【摘要】 运输脂类物质是血浆脂蛋白的基本作用。越来越多的报道显示血浆脂蛋白与微生物感染之间有重要的相互联系,血浆脂蛋白往往对微生物感染起到部分防御的作用。有大量证据说明高密度脂蛋白(high-density lipoprotein, HDL)是天然免疫的一部分,具有直接保护宿主,抗感染和炎症的作用。本研究偶然发现重组表达的M41型A群链球菌[group A Streptococcus (GAS)]的I型胶原样蛋白(streptococcal collagen-like protein 1,Scl1)能与HDL结合。Scl1是GAS表面的一种毒力因子。一些M型的GAS表达的Scl1可以与低密度脂蛋白(low-density lipoprotein, LDL)结合。为证实Scl1与HDL的相互作用,本研究从M41血清型A群链球菌(GAS)的两种菌株(MGAS6183、CMCC32198)中克隆了Scl1基因,构建了全长和不同截短的表达载体并在大肠杆菌BL21中表达和纯化。通过亲和色谱实验以及Western blot,Dot blot, ELISA实验证实了两种菌株来源的rScl1都可以通过N末端的可变区(variable regions,V区,含84个氨基酸)结合HDL,而截去V区N末端42个氨基酸或C末端42个氨基酸的rScl1,均不能与HDL结合,说明完整的V区结构在rScl1与HDL的结合中起重要作用。进一步研究发现Tween 20能抑制rScl1与HDL的结合,提示rScl1与HDL的结合可能是通过疏水相互作用。由于rScl1能同时结合HDL和LDL,因此,在制备无脂血清方面可能有潜在的用途。据我们所知,这是在国际上首次发现GAS M41的rScl1与HDL结合,但是这种HDL与GAS的相互作用在GAS致病性或机体抗感染中的作用还有待于深入的研究。目前,脂蛋白(a)[Lipoprotein(a), Lp(a)]的生理功能仍然不清,但是Lp(a)的组成成分-载脂蛋白(a)[Apo(a)],与纤溶酶原(Plasminogen,Plg)有高度的相似性,因此可以假设Lp(a)很可能与细菌表面的Plg受体结合,从而抑制Plg与细菌的结合,最终抑制细菌对Plg的激活作用,阻碍细菌生长和穿越人体组织屏障,在一定程度上起到抵抗细菌感染的作用。为验证这一假设,本研究用柱层析技术从血浆中分离制备Lp(a),并用酶联免疫吸附试验(ELISA)检测Lp(a)与金黄色葡萄球菌是否结合。结果初步证明Lp(a)可与金黄色葡萄球菌的菌株CMCC26003结合,而且一定浓度的赖氨酸类似物6-氨基己酸(20mM)可以抑制金黄色葡萄球菌与Lp(a)结合,提示血浆Lp(a)有可能通过其Kringle结构域上的赖氨酸结合位点与金黄色葡萄球菌结合。将金黄色葡萄球菌与Plg以及Lp(a)共孵育,再加入尿激酶(uPA),结果显示由于Lp(a)的加入,菌表面的纤溶酶活性降低,说明Lp(a)可抑制金黄色葡萄球菌对Plg的吸附。总之,本研究在国际上首次发现Lp(a)有可能与金黄色葡萄球菌结合并抑制金黄色葡萄球菌对Plg的吸附,这一发现初步证实了本研究室提出的Lp(a)抗感染假说,有助于更好地了解Lp(a)在抗感染中的作用,并且为防治病原细菌感染提供可能的新理念或技术手段。另外,本研究结果为本研究室提出的脂蛋白免疫学理论体系提供了支持和佐证。更多还原

【Abstract】 Plasma lipoproteins function primarily in lipid transportation. The cumulative evidence suggests a correlation between plasma lipoproteins and pathogenic infection, and plasma lipoproteins usually play an important role in preventing some infections.It is well documented that high-density lipoprotein (HDL) as part of innate immunity serves a protective role against infection and inflammation. We unexpectedly found that HDL bound to a recombinant Streptococcal collagen-like protein 1 (rScl1), which is a virulence factor on the surface of Group A Streptococcus (GAS). Interestingly, Scl1 expressed by some M-type GAS strains was recently demonstrated to bind low-density lipoprotein (LDL).To confirm the interaction of Scl1 with HDL, DNA fragment encoding the extracellular portions of Scl1 ,which were derived from two serotype M41 GAS strains (MGAS6183, CMCC32198) , were coloned into the plasmid vector pASK-IBA2.Full-length or different truncted variant of Scl1 were expressed in E. coli BL21. Purified rScl1s were immobilized onto affinity-chromatography columns and tested for the direct binding to purified HDL or HDL in plasma. SDS-PAGE ,Western blot and enzyme-linked immunosorbent assay (ELISA) revealed that both of rScl1s derived from above two strains via their variable region (V region) of 84 amino acids (84aa) could bind to HDL. However, neither N-terminal 42 aa- truncated variant nor C-terminal 42 aa-deleted variant at V region of rScl1 was not able to bind to HDL. This might imply that the entire structure of V region might be responsible for the binding. Furthermore, the rScl1-HDL binding might be mediated by hydrophobic interaction since Tween 20 inhibited it. Additionally, rScl1 might have a potential application in the production of lipid-free serum due to the dual HDL and LDL binding capabilities. To our knowledge, it is the first time to identify the binding of rScl1 to HDL. However, the pathogenic or anti-infective role of the HDL-GAS interaction remains elusive.The biological function of lipoprotein (a) [Lp(a)] is yet unclear. The apolipoprotein (a) [Apo (a)] of Lp(a) has a high similarity to plasminogen (Plg). We hypothesized that Lp(a) may bind to Plg receptor on bacterial surface, thereby inhibiting the binding of Plg to bacteria, and ultimately inhibiting the activation of bacterium-absorbed Plg by Plg activator from either host or some bacteria, as well as impeding the bacteria from passing the barriers of human tissues. To test our hypothesis, the binding between Lp (a) and S. aureus were detected by enzyme-linked immunosorbent assay , and the preliminary data showed that Lp (a) bound to the cell of S. aureus CMCC26003 strain, and the lysine analogues EACA (20mM) inhibited the binding. The results indicated that Lp (a) might through its lysine binding sites bind to S. aureus. Incubation of S. aureus with plasminogen and Lp(a) followed by uPA treatment showed the decreased plasmin activity on the surface of S.aureus when compared with the similar activiation experiment in the absence of Lp(a). These findings suggested that Lp(a) inhibited plg from recruiting by S. aureus.To our knowledge, this is the first time to demonstrate that Lp (a) might bind to S. aureus, and inhibited the recruitment of Plg by S. aureus. These findings primarily confirm our initial hypothesis and might shed light on the role of Lp(a) in preventing infections. Moreover, a potential technique in the treatment and prevention of infection in humans might be developed based on our results.In addition, these results provide the novel evidence to further support that plasma lipoproteins may be important components of the host defense system.更多还原