利用重叠PCR实现乙肝表面抗原CTL表位的替换

Replacing CTL epitope of recombinant HBsAg by SOEing PCR

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 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 peHBsAg 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 plasmid pBSSK/GPC3 and then inserted into pcDNA3. 1 ＋ vector. Results Sequencing and restrict endonulease digestion indicated that eukaryotic expression vector pcDNA- HBsAg/GPC3 was constructed successfully. Conclusion The endogenous CTL epitope LLD of HBsAg is replaced by the EYILSLEEL, and an eukaryotic expression vector pcDNA-HBsAg/GPC3 is constructed.