乙型肝炎病毒表面抗原双顺反子的真核表达载体构建

Construction of a dicistronic expression plasmid vector containing double-valent hepatitis B surface gene

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 in order to enhance vaccin response, we constructed a dicistronic expression plasmid containing double HBsAg immunogenes. Methods At first, pcDNA3.1 plasmid vector was digested with NheI and EcoRI to get the coding sequence of the small (S) surface Protein of HBV, then cloned into pCI-neo vector and named it pCI-S. By PCR amplification, the product of IRES-S was digested with Sail & BamHI, and cloned into pBluescript IIK+S to generate pBKS-IRES-S vector, then subcloned to the PCI-S plasmid to generate pCI-S-IRES-S, which is a dicistronic plasmid of double value HBsAg genes. Results Two plasmids we constructed were digested with related restriction nucleic enzymes. Sequence analysis of HBsAg and IRES-S gene did not reveal any mutation. Conclusions The construction of dicistronic plasmid of divalue HBsAg immunogenes has been well cloned, which is convenient for further research on cell expression and gene immunization in animals.