携改良rHBVpre-S1基因酵母表达载体的构建及其免疫原性鉴定  

Construction of yeast expression vector carrying recombinant HBVpre-S1 gene and identification of its immunogenicity

作　　者：夏冬[1] 甘云辉[1] 姚晖[1] 徐亮[1] 

机构地区：[1]泸州医学院附属医院普外科,四川泸州646000

出　　处：《现代医学》2013年第4期221-225,共5页Modern Medical Journal

基　　金：四川省卫生厅科技项目(090210);泸州市科技局重点科技项目(2009-S-16)

摘　　要：目的:构建携改良rHBVpre-S1基因的酵母表达载体,探讨rHBVpre-S1蛋白的免疫原性。方法:以我国HBV流行株adr的DNA为模板,PCR法扩增HBVpre-S1基因,再应用PCR定点突变技术对HBVpre-S1的抗原决定簇氨基酸的编码基因作无免疫原性修饰,酶切后与酵母穿梭表达载体pGBKT7连接,测序后命名为pGBKT7-rHBVpre-S1,提取转化质粒的酵母蛋白行免疫蛋白印迹分析。蛋白纯化后,以蛋白颗粒行腹腔内注射免疫昆明小鼠,间接ELISA法检测小鼠血清中抗pre-S1抗体的阳性率。结果:成功地构建了携改良rHBVpre-S1基因的酵母表达载体,免疫印迹检测出人rHBVpre-S1蛋白的表达,昆明小鼠经目的抗原免疫5次后其抗体阳性率显著性低于对照组。结论:构建的pGBKT7-rHBVpre-S1基因酵母表达载体兼具嗜肝性及无免疫原性的优势,可为肝病的靶向基因治疗提供理想的载体。Objective： To construct a yeast expression vector carrying recombinant rHBV pre-S1 gene and to investigate its immunogenicity. Methods ： By using the DNA of HBV strain adr as a template, PCR was performed to amplify HBV pre-S1 gene and the antigenic determinant amino acid of the PCR product was modified by sitedirected mutagenesis method. Then the mutated HBVpre-S1 gene was digested and coloned into the shuttle vector pGBKT7. The resulting pGBKT7-rHBVpre-S1 was transformed into yeast cell AH109 by lithium acetate method, the yeast protein was isolated and analyzed by Western bloting. Ten Kunming （KM） mice were given intraperitoneal injections of purified target protein, and the anti-preS1 antibody elicited rate was detected by indirect ELISA. Results： The yeast expression vector of modified HBV pre-S1 gene was constructed successfully, and the target protein was detected by Western bloting. After 5 rounds of immunization, the positive rate of antipreSl antibody in the sera of target KM mice was absolutely lower compared with the control group. Conclusion：Construction of recombinant yeast expression vector carrying modified rHBVpre-S1 gene, with liver-directed and without immunogenicity concurrently, may serve as an ideal vector for future application in liver gene therapy.

关 键 词：乙型肝炎病毒 pre-S1基因 免疫原性 酵母表达载体 

分 类 号：R512.62[医药卫生—内科学]