恶性疟原虫MSP1蛋白C-末端19kD蛋白在家蚕杆状病毒表面展示系统中的表达研究

发布时间：2018-01-29 18:03

本文关键词： 恶性疟原虫MSP1蛋白家蚕杆状病毒表面展示技术重组杆状病毒BmNPV-gp64-MSP1_(19c)　出处：《浙江理工大学》2012年硕士论文　论文类型：学位论文

【摘要】：疟疾（Malaria）是一种由疟原虫造成的，以按蚊为主要媒介传播的全球性急性寄生虫传染病。疟疾的临床症状主要发生在疟原虫裂殖子孢子在人体红细胞内无性增殖阶段，目前疟疾疫苗研究的热点主要是基于阻止此阶段疟原虫的无性裂殖来预防疟疾的感染和发病。恶性疟原虫裂殖子表面膜蛋白1C-末端的19kD片段（PfMSP1_(19C)）在裂殖子孢子侵染红细胞后仍然存在于红细胞表面，可诱导机体T细胞和B细胞产生免疫应答，因此是当前疟疾疫苗研制的重要候选抗原位点。家蚕杆状病毒表面展示技术是近几年发展起来的一种新的真核展示技术，通过将外源基因与家蚕杆状病毒的糖蛋白基因gp64融合可实现目的蛋白在病毒囊膜上的表面展示。该技术在单、多克隆抗体的制备、新疫苗的研制等领域已成为研究热点。本研究通过将MSP1_(19c)的基因片段连接到载体pFastBac1-gp64中，成功构建了真核供体质粒pFastBac1-gp64-MSP1_(19c)，该供体质粒转化E. coli DH10Bac感受态细胞后与家蚕Bacmid发生转座得到Bacmid-gp64-MSP1_(19c)。用该Bacmid-gp64-MSP1_(19c)通过脂质体介导法转染家蚕BmN细胞，在细胞内经过装配形成重组杆状病毒BmNPV-gp64-MSP1_(19c)并复制扩增获得大量重组病毒。通过PCR检测到MSP1_(19c)基因在家蚕BmN细胞中得到转录表达。用第三代BmNPV-gp64-MSP1_(19c)病毒以中等感染复数(MOI=10)接种家蚕蛹，7天后收集发病蚕蛹。Western Blotting及双向电泳检测表明重组杆状病毒在蚕蛹中表达了MSP1_(19c)蛋白，证明重组杆状病毒BmNPV-gp64-MSP1_(19c)重组成功。纯化重组病毒粒子免疫新西兰雄兔制备血清抗体，抗体经Protein A抗体纯化柱纯化后，通过ELISA检测发现抗体效价达到1:8000以上，表明该重组的病毒能有效刺激机体产生抗体。本研究证实了利用家蚕杆状病毒表面展示技术可有效将恶性疟原虫MSP1_(19c)蛋白展示在家蚕杆状病毒囊膜表面，并且该重组病毒作为免疫原能够刺激机体产生抗体和免疫细胞，，这为一种全新的疟疾疫苗的研制奠定了基础。
[Abstract]:Malaria (Malaria) is caused by Plasmodium. A global acute parasitic infectious disease with Anopheles mosquitoes as the main vector. The clinical symptoms of malaria mainly occur in the stage of asexual proliferation of Plasmodium parasite merozoite spores in human erythrocytes. At present, the focus of malaria vaccine research is to prevent malaria infection and disease by preventing the asexual cracking of Plasmodium falciparum. The 19kD fragment of merozoite surface membrane protein 1C- terminal of Plasmodium falciparum (. PfMSP1C) still exists on the surface of erythrocytes after merozoite spores infecting red blood cells. It can induce immune response of T cells and B cells. Therefore, it is an important candidate antigen site for malaria vaccine development at present. The surface display technology of Bombyx mori baculovirus is a new eukaryotic display technology developed in recent years. By fusion of foreign gene and glycoprotein gene gp64 of Bombyx mori baculovirus, the target protein can be displayed on the surface of virus envelope. The development of new vaccines has become a research hotspot. In this study, the gene fragment of MSP1 / 19c was ligated into the vector pFastBac1-gp64. The eukaryotic donor plasmid pFastBac1-gp64-MSP1 / 19c was successfully constructed. The donor plasmid was transformed into E. coli DH10Bac competent cells and transposed with silkworm Bacmid to obtain Bacmid-gp64-MSP1 / 19c). The BmN cells of silkworm, Bombyx mori, were transfected with Bacmid-gp64-MSP1 (19c) by liposome-mediated method. Recombinant baculovirus BmNPV-gp64-MSP1 (19c) was assembled in the cell, and a large number of recombinant viruses were obtained by replicating and amplifying. MSP1 / 19c was detected by PCR. The gene was transcribed and expressed in silkworm BmN cells. The third generation of BmNPV-gp64-MSP1 (19c) virus was inoculated into silkworm pupae with moderate infection of plural moi 10). After 7 days, the infected silkworm pupae was collected. Western Blotting and two dimensional electrophoresis analysis showed that the recombinant baculovirus expressed MSP1 tipp19c protein in silkworm pupae. The recombinant baculovirus BmNPV-gp64-MSP1 (19c) was successfully recombined. The purified recombinant virus particles were immunized with New Zealand male rabbits to prepare serum antibodies. The antibody was purified by Protein A antibody column, and the titer of antibody was over 1: 8000 by ELISA detection, which indicated that the recombinant virus could effectively stimulate the body to produce antibody. This study confirmed that the protein of Plasmodium falciparum MSP1 / 19c could be effectively displayed on the membrane of Bombyx mori baculovirus by using the surface display technique of Bombyx mori baculovirus. As an immunogen, the recombinant virus can stimulate the production of antibodies and immune cells, which lays the foundation for the development of a new malaria vaccine.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】