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目的利用双顺反子元件构建含 ＨＢｓＡｇ双体基因的真核表达载体，以增强 ＤＮＡ疫苗的免疫疗效。方法我们首先酶切ｐｃＤＮＡ３．１－Ｓ获得目的基因ＨＢＳＡｇ片段，将其克隆到ｐＣＩ－ｎｅｏ载体得到ｐＣＩ－Ｓ表达载体；另外通过ＰＣＲ扩增获得目的基因ＩＲＥＳ－Ｓ并定向克隆到ｐＢｌｕｅｓｃｒｉｐｔ Ｋ＋Ｓ载体，相应酶切后再次克隆到ｐＣＩ－Ｓ得到两价ＨＢｓＡｇ真核表达载体ｐＣＩ－Ｓ－ＩＲＥＳ－Ｓ，并进行酶切图谱分析和测序分析。结果ｐＣＩ－Ｓ－ＩＲＥＳ—Ｓ经相应酶切电泳显示 ７４０ ｂｐ和 １３５０ ｂｐ左右的目的基因片段，经测序鉴定， ＨＢｓＡｇ与 ＧｅｎｅＢａｎｋ ＨＢｓＡｇ ａｄｗ亚型相符，ＩＲＥＳ－Ｓ亦无任何变异。结论乙型肝炎表面抗原双体的双顺反子的真核表达载体构建成功，为在真核细胞的高效表达和基因治疗作了必要的准备。
Objective To construct a eukaryotic expression vector containing HBsAg gene using bicistronic elements to enhance the immunotherapy efficacy of DNA vaccine. Methods We first digested pcDNA3.1-S to obtain the target gene HBSAg fragment, and cloned it into pCI-neo vector to obtain pCI-S expression vector. In addition, the target gene IRES-S was amplified by PCR and cloned into pBluescript K + After corresponding digestion, cloned into pCI-S again to obtain bivalent HBsAg eukaryotic expression vector pCI-S-IRES-S, and digested map analysis and sequencing analysis. Results The corresponding fragments of 740 bp and 1350 bp were confirmed by restriction enzyme digestion. The sequence of HBsAg was consistent with the HBsAg adw subtype of GeneBank, and there was no variation of IRES-S. Conclusion The bicistronic eukaryotic expression vector of hepatitis B surface antigen (CDS) diabodies has been successfully constructed and is necessary for the efficient expression of eukaryotic cells and gene therapy.