鼠疫菌天然F1与重组rV270抗原的制备及其免疫保护作用的研究

发布时间：2018-07-05 15:32

本文选题：鼠疫菌 + 天然F1抗原　；参考：《中国人民解放军军事医学科学院》2008年硕士论文

【摘要】： 背景:鼠疫是由鼠疫耶尔森氏菌(Yersinia pestis) (以下简称鼠疫菌)引起的烈性传染病,同时也是一种标准的生物战剂和生物恐怖剂。鼠疫菌的主要保护性抗原是F1(荚膜抗原)和LcrV(V-抗原),以保护性抗原作为亚单位疫苗成为当今鼠疫疫苗的重要研究方向。F1抗原由鼠疫菌pMT1质粒上的F1操纵子编码,以多聚体的形式在细胞表面形成荚膜,F1抗体通过阻断F1蛋白的抗吞噬作用从而具有免疫保护作用。由pCD1质粒编码的V抗原(LcrV)是鼠疫菌重要的毒力因子、低钙反应调节因子和保护性抗原,但存在一定的免疫抑制作用:V抗原在体内除抑制前炎性细胞因子(γ-干扰素、肿瘤坏死因子)的释放外还增加白细胞介素-10的释放,从而抑制宿主的天然免疫应答。最近在V抗原的研究中发现产生免疫抑制作用的区段为271-300氨基酸,从全长V抗原(326个氨基酸)中去除这个区段可有效地降低V抗原的免疫抑制作用而同时保留其免疫保护作用。近来我们发现在云南感染东方型鼠疫菌的病人血清中检测不到V抗原的抗体,这也决定了采用F1和V抗原联合应用成为鼠疫疫苗的亚单位疫苗的热点和方向。方法:采用玻璃珠处理、饱和硫酸铵沉淀和分子筛过滤相结合的方案,从鼠疫菌减毒活疫苗株(EV76)的培养物中提取天然表达的F1抗原。依据已知的LcrV抗原核苷酸序列,避开LcrV抗原产生免疫抑制作用的区段设计引物,克隆表达rV270抗原。表达产物先后经过Co2+亲和层析和Sephacryl S-200 HR凝胶柱纯化,并在纯化过程中应用凝血酶切除His标签。纯化的蛋白用免疫印记法、N-末端测序、质谱肽图、高效液相色谱(HPLC)及紫外光谱扫描方法鉴定其性质和纯度。使用氢氧化铝佐剂分别吸附两种抗原后免疫BALB/c小鼠,使用一次免疫和两次免疫观察两种抗原的单一及联合免疫保护作用,从而得出抗原配伍最佳剂量和联合抗原免疫方法对比结果。结果:本试验建立了一种新型天然鼠疫菌F1抗原的提取与纯化方法,成功提取出纯度超过98%的天然F1抗原。纯化的蛋白通过免疫印记法、N-末端测序、质谱肽图、高效液相色谱(HPLC)及紫外光谱扫描方法鉴定,证明该蛋白正是鼠疫菌F1抗原。通过ELISA检测发现纯化出的天然F1抗原对F1抗体检测的敏感度高于重组rF1抗原。rV270基因成功地克隆到pET24a载体中,并以可溶性方式表达。应用Co2+亲和层析柱和Sephacryl S-200 HR凝胶柱结合凝血蛋白酶切除His标签的方法可得到不含标签的较高纯度目的蛋白。动物实验证实纯化的天然F1抗原和重组的rV270抗原单抗原及联合抗原免疫均产生较高的抗体滴度和均有良好的免疫保护作用,且得到免疫方案和抗原免疫优化剂量组,为下一步亚单位疫苗研制奠定基础。结论:1.本实验所提供的鼠疫菌天然F1抗原的提取、纯化方法,是先从鼠疫菌培养物中分离培养上清和菌体沉淀,再利用饱和硫酸铵沉淀从培养上清中以及利用玻璃珠从菌体沉淀中分别提取出各部分天然F1抗原粗蛋白,再将各部分天然F1抗原粗蛋白各自通过饱和硫酸铵沉淀、凝胶过滤得到各部分纯化的鼠疫菌天然F1抗原,保持了蛋白的天然构象,并且避免了有机溶剂对蛋白活性的影响和对环境的污染。2.为了制备不带任何载体序列的rV270抗原,采用融合表达带His标签的蛋白,然后凝血酶切除标签的策略,使目的蛋白不含有任何载体序列,保持天然的蛋白构象和生物学活性。本研究利用金属螯合亲和层析柱将带有His标签的目的蛋白与其它大量杂蛋白分离,同时利用凝血酶在亲和层析柱上完成标签的切割,然后再利用分子筛进一步对蛋白进行纯化得到了高纯和度的目的蛋白,为下一步动物免疫观察抗原的免疫保护效果提供基础。3.动物实验实验采用不同剂量组的抗原免疫,旨在寻找最佳剂量配伍来免疫动物从而刺激机体产生更高的抗体滴度,增强免疫保护作用和延长免疫时间;使用单抗原和联合抗原免疫,目的是比较单抗原和联合抗原免疫效果,结果证实联合抗原免疫较单抗原免疫能产生更高的抗体滴度;使用一次免疫方案和初次免疫后加强免疫的两次免疫方案对比,得出两次免疫较一次免疫能产生更高的抗体滴度且产生抗体滴度时间更长。从这三方面结论得到免疫方案及抗原免疫优化剂量组,为下一步亚单位疫苗的研制奠定基础。
[Abstract]:Background: plague is a strong infectious disease caused by the Yersinia pestis (Yersinia pestis). It is also a standard biological warfare agent and bioterrorism agent. The main protective antigen of Yersinia pestis is F1 (capsule antigen) and LcrV (V- antigen), and the protective antigen is used as subunit vaccine to become the plague vaccine. The important research direction.F1 antigen is encoded by the F1 operon on the pMT1 plasmid of Yersinia pestis, which forms a capsule on the cell surface in the form of polymer. The F1 antibody has the immune protection by blocking the anti phagocytosis of the F1 protein. The V antigen (LcrV) encoded by the pCD1 plasmid is an important virulence factor of the Yersinia pestis, and the low calcium response regulator and the regulation factor. Protective antigen, but there is a certain immune inhibitory effect: V antigen in the body in addition to inhibiting the release of pro-inflammatory cytokines (interferon, tumor necrosis factor) in addition to increase the release of interleukin -10, thus inhibiting the host's natural immune response. Recently, the area of the immunosuppressive effect of the V antigen was found to be 271-300. The removal of this section from the full length V antigen (326 amino acids) can effectively reduce the immunosuppressive effect of V antigen and retain its protective effect. Recently, we found that the antibodies against V antigen in the sera of Yunnan infected with the Oriental Yersinia pestis have been found to be combined with F1 and V antigens. The hotspots and directions of subunit vaccines for vaccination.
Methods: using the scheme of glass bead treatment, saturated ammonium sulfate precipitation and molecular sieve filtration, the naturally expressed F1 antigen was extracted from the culture of Yersinia pestis live attenuated vaccine strain (EV76). According to the known nucleotide sequence of LcrV antigen nucleotides, the primers were designed to avoid the immune inhibition effect of LcrV antigen, and the expression of rV270 antigen was cloned. The products were purified by Co2+ affinity chromatography and Sephacryl S-200 HR gel column, and the His tags were excised by thrombin in the purification process. The purified protein was determined by immunoimprinting, N- terminal sequencing, mass spectrometry peptide, high performance liquid chromatography (HPLC) and UV spectrum scanning method to determine its properties and purity. The BALB/c mice were immunized with two antigens. The single immunization and two immunization were used to observe the single and combined protective effects of two antigens. The results were compared with the best antigenic dosage and the combined antigen immunization.
Results: a new method for the extraction and purification of a new type of natural Yersinia pestis F1 antigen was established, and the natural F1 antigen with purity of more than 98% was successfully extracted. The purified protein was identified by immunoimprinting, N- terminal sequencing, mass spectrometry peptide, HPLC and UV spectral scanning, which proved that the protein was the F1 antigen of Yersinia pestis. ELISA detection showed that the sensitivity of the purified natural F1 antigen to F1 antibody was higher than that of the recombinant rF1 antigen.RV270 gene, which was successfully cloned into the pET24a vector and expressed in a soluble way. The method of using Co2+ affinity chromatography column and Sephacryl S-200 HR gel column combined with coagulation protease to excision the His label could be obtained without the label. The animal experiments confirmed that the purified natural F1 antigen, the recombinant rV270 antigen single antigen and the combined antigen immunization produced a high antibody titer and good immune protection, and the immunization scheme and the immune optimal dose group were obtained, which laid the foundation for the development of the next subunit vaccine.
Conclusion: 1. the extraction and purification of the natural F1 antigen of Yersinia pestis were isolated and cultured first from the culture of Yersinia pestis, and then using the saturated ammonium sulfate precipitation from the culture supernatant and the use of glass beads to extract the natural F1 antigen of each part of the natural antigen, and then the natural F1 of each part. The antigen crude protein was precipitated by saturated ammonium sulfate, and the natural F1 antigen of Yersinia pestis was purified by gel filtration. The natural conformation of the protein was preserved, and the effect of organic solvents on the activity of protein and the environmental pollution.2. were avoided to prepare the rV270 antigen without any carrier sequence, and the fusion expression with the His label was used. In this study, the target protein with the His label was separated from a large number of other proteins with a metal chelating affinity chromatography column, and the tag was cut on the affinity chromatography column by thrombin. And then the protein was further purified by molecular sieve to obtain the high purity and degree target protein, which provided the basis for the immune protection effect of the next animal immune observation antigen in the.3. animal experiment experiment using the antigen immunization of different dose groups, aiming at finding the best dose to match the immune animals to stimulate the organism to produce higher production. The titer of antibody, enhanced immune protection and prolongation of immune time; immunization with single antigen and combined antigen was used to compare the immune effect of single antigen and combined antigen. The results confirmed that the combined antigen immunization produced a higher antibody titer than the single antigen immunization, and the two immunization was enhanced after the first immunization program and the first immunization. The comparison of the scheme shows that the two immunization can produce a higher antibody titer and produce a longer antibody titer. From these three aspects, the immune scheme and the optimal dose group of the antigen immunization are obtained, which lays the foundation for the development of the next subunit vaccine.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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