论文标题:轮状病毒VP4、VP7抗原在烟草中的表达
The Experssion of Rotavirus VP4 and VP7 in Tobacco
论文作者 包慧芳
论文导师 陈创夫；祝建波,论文学位 硕士,论文专业 生物化学与分子生物学
论文单位 石河子大学,点击次数 648,论文页数 55页File Size4439k
2005-05-01论文网 http://www.lw23.com/lunwen_318427/ 轮状病毒;VP4;VP7;转基因植物;植物疫苗
Rotavirus; VP4; VP7; transformed plant; plant vaccine
轮状病毒(Rotavirus,RV)是世界范围内引起婴幼儿和各种幼龄动物病毒性腹泻的主要病原体,RV感染在社会中引起严重的经济损失。迄今,对RV感染除了对症处理外并无特效药物,因此研究和开发安全有效的轮状病毒疫苗具有很重要的意义。研究表明,将细菌性和病毒性病原体抗原的编码基因导入植物细胞,能够表达出较好保留天然免疫原性的抗原,这为利用转基因植物生产疫苗提供了可靠的依据。VP4和VP7是轮状病毒的主要外壳蛋白和中和抗原,因此,将VP4和VP7基因在植物中表达,获得具有免疫原性的轮状病毒植物口服疫苗,具有潜在的社会意义和经济价值。 本研究以VP4和VP7为目的基因,设计具有BamH Ⅰ和Sac Ⅰ酶切位点及Kozak序列的VP4基因引物,采用套叠PCR对VP4进行定点突变,构建了pBI121-VP4植物表达载体;同时设计具有BamH Ⅰ和Sal 1酶切位点及Kozak序列的VP7基因引物,构建了pBin438-VP7植物表达载体。利用农杆菌介导法,转化烟草叶盘,经卡那霉素(Kan)多重筛选,获得抗性植株。对抗性植株提取DNA进行PCR分析,40-50%抗性植株呈阳性。选取几株生长良好的PCR检测为阳性的转基因烟草植株提取总DNA经HindⅢ内切酶消化后进行Southen-blot分析,结果表明所有待检样品均出现较强的特异杂交信号,对Southern-blot检测为阳性的植株,提取总蛋白进行Western-blot分析,证明部分植株表达了轮状病毒VP4和VP7。 本研究为研究和开发新型轮状病毒口服疫苗提供一条新的途径和方法,具有一定的理论意义和应用前景。
Rotavirus (RV) is the major pathogen causing Virus-based diarrhea in infants and young animals of the world, which brings serious damages to economy of the world. Up to the present,there is no effective medicine for the allopathy, therefore, it is necessary to develop an effective rotavirus vaccine.It is suggested that foreign antigen proteins with immunogenicity can be effectively expressed if the antigens from infectious bacteria or viruses are introduced into plants,which provides a good approach to producing vaccine by transgenic plants.VP4 and VP7 are the major capsule protein and neutralized antigens. Therefore, introducing VP4 and VP7 genes into plant to develop edible plant vaccine of rotavirus would provid potential meaning for society and economy.In this study, the VP4 and VP7 genes were amplified and modified by PCR. The kozak sequence was placed upstream of the initiation codons of VP4 and VP7, which was advantageous for the gene expression. And the VP4 gene was mutated in a certain point by PCR.By inserting the modified VP4、 VP7 gene of rotavirus into plant expression vector pBI121,pBin438 respectively,2 expression plasmids pBI121-VP4 and pBin438-VP7 were constructed and then transformed to Agrobacterium EHA105 directly. Then pBI121-VP4 and pBin438-VP7 were introduced into tobacco leaf disc. Transformed shoots were selected on solid medium containing Kanamycin. These transformed shoots were checked by PCR, Southern-blot and Western-blot.The Southem-blot indicated that most of transformed shoots had the interested gene integrated into the genome of tobacco, but only few transformed shoots were positive in Western-blot assay.This research provided a new method for new rotavirus vaccine, and presented theorical significance and applicative prospect.
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