本文關(guān)鍵詞： 惡性瘧原蟲MSP1蛋白 家蠶桿狀病毒表面展示技術(shù) 重組桿狀病毒BmNPV-gp64-MSP1_(19c)　出處：《浙江理工大學》2012年碩士論文　論文類型：學位論文

【摘要】：瘧疾（Malaria）是一種由瘧原蟲造成的，以按蚊為主要媒介傳播的全球性急性寄生蟲傳染病。瘧疾的臨床癥狀主要發(fā)生在瘧原蟲裂殖子孢子在人體紅細胞內(nèi)無性增殖階段，目前瘧疾疫苗研究的熱點主要是基于阻止此階段瘧原蟲的無性裂殖來預防瘧疾的感染和發(fā)病。惡性瘧原蟲裂殖子表面膜蛋白1C-末端的19kD片段（PfMSP1_(19C)）在裂殖子孢子侵染紅細胞后仍然存在于紅細胞表面，可誘導機體T細胞和B細胞產(chǎn)生免疫應(yīng)答，因此是當前瘧疾疫苗研制的重要候選抗原位點。家蠶桿狀病毒表面展示技術(shù)是近幾年發(fā)展起來的一種新的真核展示技術(shù)，通過將外源基因與家蠶桿狀病毒的糖蛋白基因gp64融合可實現(xiàn)目的蛋白在病毒囊膜上的表面展示。該技術(shù)在單、多克隆抗體的制備、新疫苗的研制等領(lǐng)域已成為研究熱點。 本研究通過將MSP1_(19c)的基因片段連接到載體pFastBac1-gp64中，成功構(gòu)建了真核供體質(zhì)粒pFastBac1-gp64-MSP1_(19c)，該供體質(zhì)粒轉(zhuǎn)化E. coli DH10Bac感受態(tài)細胞后與家蠶Bacmid發(fā)生轉(zhuǎn)座得到Bacmid-gp64-MSP1_(19c)。用該Bacmid-gp64-MSP1_(19c)通過脂質(zhì)體介導法轉(zhuǎn)染家蠶BmN細胞，在細胞內(nèi)經(jīng)過裝配形成重組桿狀病毒BmNPV-gp64-MSP1_(19c)并復制擴增獲得大量重組病毒。通過PCR檢測到MSP1_(19c)基因在家蠶BmN細胞中得到轉(zhuǎn)錄表達。用第三代BmNPV-gp64-MSP1_(19c)病毒以中等感染復數(shù)(MOI=10)接種家蠶蛹，7天后收集發(fā)病蠶蛹。Western Blotting及雙向電泳檢測表明重組桿狀病毒在蠶蛹中表達了MSP1_(19c)蛋白，證明重組桿狀病毒BmNPV-gp64-MSP1_(19c)重組成功。純化重組病毒粒子免疫新西蘭雄兔制備血清抗體，抗體經(jīng)Protein A抗體純化柱純化后，通過ELISA檢測發(fā)現(xiàn)抗體效價達到1:8000以上，表明該重組的病毒能有效刺激機體產(chǎn)生抗體。 本研究證實了利用家蠶桿狀病毒表面展示技術(shù)可有效將惡性瘧原蟲MSP1_(19c)蛋白展示在家蠶桿狀病毒囊膜表面，并且該重組病毒作為免疫原能夠刺激機體產(chǎn)生抗體和免疫細胞，，這為一種全新的瘧疾疫苗的研制奠定了基礎(chǔ)。
[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.
【學位授予單位】：浙江理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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本文編號：1474014