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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,应用重叠延伸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。
Objective To construct a DNA vaccine for the prevention and treatment of hepatitis B by replacing the endogenous CTL epitope LLD in HBsAg with CTL epitope EYILSLEEL of glypican-3 (GPC3). Methods HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / HBsAg / GPC3 was amplified by PCR. The recombinant plasmid was inserted into the eukaryotic expression vector pcDNA3.1 + by DNA recombination technique. The eukaryotic expression vector was identified by PCR, restriction enzyme digestion and sequencing. The DNA vaccine expressing CTL epitope EYILSLEEL was constructed. Results The results of enzyme digestion and sequencing showed that the sequence was mismatched, and the eukaryotic plasmid pcDNA3-HBsAg / GPC3 which fully expressed the EYILSLEEL epitope was obtained. Conclusion The endogenous CTL epitope of HBsAg was successfully replaced by EYILSLEEL epitope, and the pcDNA3-HBsAg / GPC3 eukaryotic expression plasmid was constructed.