轮状病毒外壳抗原蛋白VP4基因转化苎麻的研究  

Study on the Transformation of Rotavirus Coat Epitope Protein Gene VP4 into Boechmeria nivea L.Guad

作　　者：孔华[1] 郭安平[1] 郭运玲[1] 刘恩平[1] 章霄云[1] 贺立卡[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所热带作物生物技术国家重点实验室,海南海口571101

出　　处：《安徽农业科学》2006年第24期6460-6462,6464,共4页Journal of Anhui Agricultural Sciences

基　　金：海南省自然科学基金资助(30301)

摘　　要：利用套叠PCR技术对轮状病毒外壳抗原蛋白VP4基因进行了定点突变,将改造后的基因插入pBI121构建植物表达载体。在设计PCR引物时,引入植物表达载体pBI121具有的克隆位点BamH I和Sac I,利用BamH I和Sac I切除pBI121上的GUS基因并使载体质粒线性化,用同样的2种酶消化pTVP4克隆载体获得VP4基因片段,使之形成与线性化pBI121质粒相同的粘性末端。在T4DNA连接酶的作用下,将线性化pBI121质粒和酶切后的VP4基因片段连接成新的重组质粒pBI121/VP4,通过直接转化法转化到根癌农杆菌(Agrobacteriumefaciens)EHA105菌株中,获得农杆菌工程菌株。采用叶盘转化法转化苎麻(Boehmeria.nivea L.Guad)栽培品种圆叶青,以卡那霉素抗性作为转化植株的筛选标记获得抗性植株。经PCR、PCR Southernblot分析表明,初步获得了轮状病毒外壳蛋白VP4的转基因苎麻植株。Rotavirus is one of the main pathogens which cause the virus diarrhea of young animals. The outbreak of the epidemic will cause serious financial loss. It is reported in pigs, cattle, sheep, dogs, rabbits and many kinds of fowls infected with rotavirus in our country. In this research, the gene encoding porcine rotavirus coat epitope protein VP4 was site -specific mutated by overlap extension of PCR technique. The modified gene was inserted in pBI121 to construct the plant expression vector. In order to be convenient for amplify, BamH Ⅰ and Sac Ⅰ sites were added to the two borders respectively. Gent GUS was removed from pBI 121 by enzyme BamH Ⅰ and Sac.Ⅰ, and then the vector was linearized. The cloning vector was digested with the same two enzymes and the fragment VP4 which had the same cohesive ends as the linearized pBI121 was obtained. The two fragments were connected by T4 DNA ligasc to construct recombinant plasmid pBI121/VP4. And then it was transformed to Agrobacterium tumefaciens EHA105 strain directly. A. tumefaciens EHA105 harboring pBI121/VP4 was used to infect the leaf disk of Boechmeria nivea LGuad. With kanamycin selection, the resistant plants were obtained, which was proved by PCR and PCR-Southern blot analysis. This research was useful to make further researches into the structure and function of gene VP4. It also offered a new way to research and develop oral vaccine which may change the conventional vaccine inoculation and cut down the cost of vaccine production.

关 键 词：轮状病毒 VP4基因 苎麻 

分 类 号：Q78[生物学—分子生物学]