疟疾多抗原表位基因表达载体的构建及其在烟草中的表达

Preliminary study on cholera toxin B subunit Plasmodium falciparum chimerical gene Transgenic tobacco

本文首次报道疟疾多表位抗原基因在转基因烟草中表达成功。疟疾是当今最需要研究有效疫苗的主要传染病之一。过去的研究表明，ＡＷＴＥ基因编码的疟疾多种抗原表位是有效的抗疟表位，ＣＴＢ基因编码的霍乱毒素Ｂ亚基，是一种既能引起细胞免疫又能引起体液免疫的免疫载体和佐剂。本研究把ＡＷＴＥ－ＣＴＢ融合基因构建到植物表达载体ｐＢＶＧ－ｎｙ１上，采用共转化的方法，通过基因枪导入转化烟草。经ＰＣＲ扩增ＡＷＴＥ－ＣＴＢ基因片段检测，证实了疟疾多表位抗原基因在转基因烟草中的整合。ＳＤＳ－ＰＡＧＥ蛋白电泳结果显示转基因烟草中表达了ＡＷＴＥ－ＣＴＢ融合基因分子量相同的特异蛋白。经抗原性分析实验和Ｗｅｓｔｅｒｎ免疫印迹实验结果表明，特异表达的融合蛋白可与ＣＴＢ和ＡＷＴＥ抗体结合，具有ＣＴＢ和ＡＷＴＥ抗原性。

Preliminary study on the development of CTB AWTE transgenic tobacco against Plasmodium falciparum is discussed. Multiple epitopes of P.falciparum were cloned down stream of cholera toxin B subunit gene in the same reading frame (CTB AWTE),and inserted into pBI426 to generate expression vector pBVG ny1. Particle bombardment was used to transform tobacco plant. PCR of transformed tobacco DNA demonstrated the integration of CTB AWTE expression vector into the transgenic tobacco genome. Antigenicity assay showed that the fusion protein of AWTE CTB expressed in the transgenic tobacco can combine with CTB and NKNDD(one epitope in AWTE) antibody. ELISA analysis of proteins extracted from transgenic tissues showed positive reaction to CTB antibody. Preliminary Western analysis CTB antibody indicated the presence of a polypeptide with a molecular weight similar to CTB AWTE.