炭疽杆菌S-层蛋白BA3338与炭疽毒力的关系

When we analyzed the different expression proteins of B.anthracis A16R in vitro and in vivo using rabbit model, we identified an obviously up-regulated protein in the rabbit's intestinal tract. This protein is identified by PMF spectrum as an S-layer protein BA3338 but the function of which has not been reported in the literature so far. Further researches on this protein indicated it could interact with some proteins of human blood serum, such as: Fibronectin(FN1,FN2), acute phase reaction factor(inter-alpha-trypsin inhibitor family heavy chain-related protein,IHRP) and complement regulatory protein(SP40/40). These findings indicated that the BA3338 protein may contribute to the virulence of the B.anthracis A16R..Our work of "The research on the function of the Bacillus anthracis S-layer protein BA3338" was supported by the National Natural Science Foundation of China in 2010. This project has been finished by the end of 2011. In this project, we construct an isogenic mutant that BA3338 be replaced via allelic exchange with a spectinomycin resistance cassette on base of homologus recombination. Then we compare the virulence,the capacity of spore forming,immunogenicity,growth and metabolizability and the proteins expression profile of the vaccines strain A16R and the isogenic mutant strain. The findings of our project indicate that the BA3338 gene has no effect on the growth,metabolizability and the spore forming of the B.anthracis, but the virulence of the B.anthracis was greatly attenuated when this gene was knocked out. These findings further confirmed that the BA3338 was associated with the virulence of the B.anthracis..From these findings, We are now thinking about an another issue that "How does the BA3338 protein affect the virulence of the B.anthracis?". To answer this question, We will carry out the following researches. How does the BA3338 proteins distribute on the surface of the B.anthracis A16R? What role does the BA3338 proteins play in the interaction between B.anthracis and the host cell? What is the relationship between the phase expression change of BA3338 protein and the virulence of the B.anthracis? Does BA3338 protein have an influence on the B.anthracis distribution in the organ of the infected animals. What is the relationship between the BA3338 and the two other S-layer proteins in B.anthracis in the aspect of influence the virulence of B.anthracis? We will finish the comparative proteomics analysis between the B.anthracis vaccine strain A16R and the variant strain A16R△BA3338 to verificate and analyse the biological function of the different proteins. These findings will provide a new target to improve the vaccine against the anthrax.

S-层蛋白是位于炭疽杆菌细胞壁表面的一种类晶体结构，具有许多重要的功能。本课题组前期结果显示：家兔肠内培养的炭疽菌上清样品中，S-层蛋白BA3338蛋白表达显著上调，且与人血清中纤连蛋白、急性期反应蛋白、补体调节蛋白等存在相互作用。这说明：BA3338可能与炭疽菌的致病性有关。课题"炭疽杆菌S-层蛋白BA3338功能研究"的结果显示：BA3338缺失不影响炭疽菌的生长代谢及芽胞形成，却使毒力显著减弱，进一步确认了BA3338与炭疽菌毒力之间的关系。.本研究拟从结构分布、在炭疽菌与宿主细胞粘附过程中的作用、与宿主细胞相互作用蛋白的筛选、对炭疽菌在感染动物体内分布的影响、与其它S-层蛋白在影响炭疽杆菌毒力方面的关系及比较蛋白质组学研究，深入分析BA3338蛋白是如何影响炭疽菌的毒力的。这些结果不仅能够拓宽人类对炭疽杆菌致病机理的认识，而且将为疫苗株A16R的改进提供了一个很好的靶标。

S-层蛋白是位于炭疽杆菌细胞壁表面的一种类晶体结构，具有许多重要的功能。本课题组前期结果显示：家兔肠内培养的炭疽菌上清样品中，S-层蛋白BA3338蛋白表达显著上调，且与人血清中纤连蛋白、急性期反应蛋白、补体调节蛋白等存在相互作用。这说明：BA3338可能与炭疽菌的致病性有关。课题“炭疽杆菌S-层蛋白BA3338功能研究”的结果显示：BA3338缺失不影响炭疽菌的生长代谢及芽胞形成，却使毒力显著减弱，进一步确认了BA3338与炭疽菌毒力之间的关系。.本研究对BA3338蛋白进行了原核表达，制备了多克隆抗体；通过免疫荧光方法，研究了BA3338蛋白在炭疽菌表面的分布，显示BA3338蛋白均匀分布于炭疽杆菌整个表面；通过流式细胞技术，研究了BA3338蛋白的相表达情况，结果显示，BA3338蛋白在对数期时表达丰度较高，而在平台期和衰亡期是表达量减少；通过小鼠攻毒实验研究，发现BA3338蛋白缺失使炭疽杆菌的毒力减弱，并且细菌的器官分布发生了变化。通过比较蛋白质组学研究筛选到23个差异蛋白，其中11个为上调，12个下调。