HBc病毒样颗粒在筛选多功能单克隆抗体中的应用研究

发布时间：2018-03-17 06:04

本文选题：乙肝病毒核心蛋白病毒样颗粒(HBc-VLP)　切入点：病毒样颗粒(VLP)　出处：《中南大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:乙肝病毒核心蛋白可自我组装成病毒样颗粒(VLP),HBc-VLP作为表位递呈系统可用于携带外源表位。除了应用于VLP疫苗和表位疫苗以外,VLP还可用于病毒颗粒的摄取,病毒药物活性以及树突状细胞(DCs)的抗原递呈功能等研究中。然而,我们发现一些商业化单克隆抗体并不能特异性识别HBc-VLP,同样也无法应用于细胞内病毒样颗粒的定位研究,于是我们不得不购买大量单抗以找到一种合适的,这一过程无疑费力又费钱。因次,要制备出一个HBc-VLP特异性多功能单克隆抗体以满足多种研究的不同需要。方法:①构建与表达HBV核心抗原:截短的HBc基因(aa1-144)通过PCR扩增而来,PCR产物经酶切后,转入表达载体pET-28a。阳性质粒转染BL21(DE3)大肠杆菌菌株,用IPTG诱导;②快速纯化重组HBc蛋白:离心获得湿重为9克的菌体,重悬加入溶菌酶后超声破菌获得包含体。将包含体加入10ml变性缓冲液(50mM Tris-HCL,pH 8.0和0.1M尿素),不能溶解的成分通过离心(30 min 12000g)去除。用复性缓冲液稀释至2mg/ml的浓度。复性蛋白溶液用双蒸水(pH6.0)透析以去除残留的尿素。并用SDS-PAGE确定其纯度;③电镜分析所制备的VLP;④制备抗HBc-VLP单克隆抗体:用HBc-VLP(aa1-144)作为免疫原来免疫BALB/c雌鼠。经过几次免疫后,将抗体滴度高的BALB/c鼠的脾细胞与SP2/0骨髓瘤细胞融合。先用ELISA筛选,再进行western印迹分析以进一步筛选,获得HBc-VLP特异性抗体;⑤鼠单核-巨噬细胞系RAW264.7的吞噬作用的检测;⑥利用共聚焦激光扫描显微镜对HBc-VLP表位疫苗的摄取过程进行观察;⑦HepG2.2.15细胞中活HBV的观察:将HepG2.2.15细胞接种于玻片上并培养,以观察先前所筛选的阳性单抗是否能与自然病毒颗粒反应。HepG2细胞作为阴性对照。结果:①成功构建了pET-28a-HBc(aa1-144),经SDS-PAGE分析,其所表达的目的蛋白分子量为19kDa;②经western印迹分析验证该目的蛋白的确具备HBc抗原的免疫原性;③0.1M尿素所溶解的HBc蛋白可形成病毒样颗粒;经超纯水透析后的HBc-VLP的结构更加规则统一;④用HBc-VLP作为免疫原来免疫BALB/c雌鼠通过ELISA和western印迹分析筛选到5个HBc-VLP特异性抗体;⑤筛选到3个可与被APC摄取的HBc-VLP相结合的特异性单抗。这三个单抗的编号分别为3H2D7-F6,1H7D7-D2和388E6-D3;⑥利用HepG2.2.15细胞系筛选到1个单抗能特异性结合自然状态下的HBc颗粒,此单抗编号为3H2D7-F6。结论:①以截短的HBc-VLP骨架作免疫原,以此表位递呈系统为筛选平台,最终筛选出1个可用于鉴定HBc-VLP,嵌合体HBc-VLP疫苗和细胞内乙肝病毒衣壳蛋白的抗体。该抗体具备多功能抗HBc-VLP作用,应用此多功能单抗可节约大量经费。 ②构建与表达的目的蛋白具有HBc抗原的免疫原性。 ③0.1M尿素所溶解的HBc蛋白可形成病毒样颗粒。
[Abstract]:Objective: HBV core protein can be self-assembled into virus-like particles HBc-VLP as epitope presenting system can be used to carry exogenous epitopes. It can also be used for the uptake of viral particles in addition to VLP vaccine and epitope vaccine. However, we found that some commercial monoclonal antibodies can not specifically recognize HBc-VLP, nor can they be applied to the localization of virus-like particles in cells. Therefore, we have to purchase a large number of McAbs to find a suitable one, and this process is painstaking and costly. Therefore, we have to produce a HBc-VLP specific multifunctional monoclonal antibody to meet the different needs of many kinds of research. Methods HBV core antigen: truncated HBc gene was amplified by PCR. The product was digested into the expression vector pET-28a. the positive plasmid was transfected into E. coli strain BL21mDE3. The recombinant HBc protein was rapidly purified by IPTG induction. The recombinant HBc protein was obtained by centrifugation with a wet weight of 9 g. After adding lysozyme in heavy suspension, the inclusion was obtained by ultrasonic breaking. The inclusion body was added to 10ml denaturing buffer solution (50mm Tris-HCL, pH 8.0 and 0.1M urea). The insoluble components were removed by centrifugation (30 min 12000g). The refolding buffer was diluted to the concentration of 2mg / ml. Sex protein solution was dialyzed with double distilled water pH 6.0) to remove residual urea. Monoclonal antibody against HBc-VLP was prepared by using SDS-PAGE to determine its purity. Monoclonal antibody against HBc-VLP was prepared by using HBc-VLPna1-144). After several times of immunization, the female mice were immunized. The spleen cells of BALB/c mice with high antibody titer were fused with SP2/0 myeloma cells. The spleen cells were screened by ELISA and then analyzed by western blotting. Detection of phagocytosis of monocyte macrophage RAW264.7 derived from HBc-VLP specific antibody the uptake of HBc-VLP epitope vaccine in 7HepG2.2.15 cells was observed by confocal laser scanning microscopy (confocal laser scanning microscope). Planted on a glass and cultured, To observe whether the previously screened positive monoclonal antibody can react with natural virus particles. HepG2 cells were used as negative control. Results the pET-28a-HBcca1-144G was successfully constructed by 1: 1. The molecular weight of the expressed protein was 19kDaNi2 by SDS-PAGE analysis. The result of western blot analysis showed that the target protein had the immunogenicity of HBc antigen and the HBc protein dissolved by 30.1 M urea could form virus-like particles. After ultrapure water dialysis, the structure of HBc-VLP was more regular and uniform. HBc-VLP was used to immunize BALB/c female mice. Five HBc-VLP specific antibodies were screened by ELISA and western blotting analysis. Three of them could be combined with HBc-VLP uptake by APC. The numbers of the three McAbs were 3H2D7-F6H7D7-D2 and 388E6-D3O6, respectively, which were screened by HepG2.2.15 cell line to specifically bind to HBc particles in natural state. The McAb number is 3H _ 2D _ 7-F _ 6. Conclusion the truncated HBc-VLP skeleton was used as the immunogen by using the epitope presenting system as the screening platform. Finally, an antibody which can be used to identify HBc-VLP, chimeric HBc-VLP vaccine and hepatitis B virus capsid protein was screened. The antibody has multifunctional anti HBc-VLP effect, and the application of this multifunctional monoclonal antibody can save a lot of money. 2 the constructed and expressed target protein has immunogenicity of HBc antigen. HBc protein dissolved in 30.1 M urea can form virus-like particles.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【共引文献】