| 利用重叠PCR实现乙肝表面抗原CTL表位的替换 |
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| 引用本文： | 王文敬,黎诚耀,李明松.利用重叠PCR实现乙肝表面抗原CTL表位的替换[J].第三军医大学学报,2009,31(7). |
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| 作者姓名： | 王文敬 黎诚耀 李明松 |
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| 作者单位： | 1. 南方医科大学,生物技术学院输血医学系,广州,510515
2. 南方医院消化科,广州,510515 |
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| 基金项目： | 国家重点基础研究发展规划(973计划)，广东省自然科学基金，广东省科技攻关计划 |
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| 摘 要： | 目的 以磷酸酰肌醇蛋白聚糖-3蛋白(glypican-3,GPC3)的CTL表位EYILSLEEL替换HBsAg中内源性CTL表位LLD,构建用于预防和治疗乙肝的DNA疫苗.方法 以重组表面抗原基因pcHBsAg质粒为模板,应用重延伸PCR扩增出含EYILSLEEL表位的HBsAg基因HBsAg/GPC3,将其插入pBSSK+载体中,构建出pBSSK/GPC3载体.利用DNA重组技术将其定向插入真核表达载体pcDNA3.1+中,将真核表达载体经PCR、酶切和测序鉴定,构建表达CTL表位EYILSLEEL的DNA疫苗.结果 酶切和测序的结果显示序列无错配,获得了完整表达EYILSLEEL表位的真核质粒pcDNA3-HBsAg/GPC3.结论 成功将EYILSLEEL表位替换HBsAg的内源性CTL表位,构建了pcDNA3-HBsAg/GPC3真核表达质粒.
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| 关 键 词： | 表位替换 磷酸酰肌醇蛋白聚糖-3蛋白 细胞毒T淋巴细胞表位 重叠延伸PCR |
Replacing CTL epitope of recombinant HBsAg by SOEing PCR |
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| WANG Wen-jing,LI Cheng-yao,LI Ming-song.Replacing CTL epitope of recombinant HBsAg by SOEing PCR[J].Acta Academiae Medicinae Militaris Tertiae,2009,31(7). |
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| Authors: | WANG Wen-jing LI Cheng-yao LI Ming-song |
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| Institution: | WANG Wen-jing1,LI Cheng-yao1,LI Ming-song2(1Faculty of Blood Transfusion,School of Biotechnology,2Department of Gastroenterology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China) |
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| Abstract: | Objective To replace the endogenous CTL epitope LLD in HBsAg with EYILSLEEL of glypican(GPC3)in order to prepare a GPC3-HBsAg multiple peptides vaccine for HBV infection.Methods The HBsAg/GPC3 DNA was amplified by gene splicing by overlap extension(SOEing)PCR from pcHBsAg plasmid and then inserted into pBSSK+ vector to construct a pBSSK/GPC3 vector.The vector was then identified by PCR,double digesting and sequencing.The fragment encoding GPC3 CTL epitope EYILSLEEL was obtained by double digesting the plasm... |
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| Keywords: | epitope shift glypican-3 CTL epitope SOEing PCR |
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