乙型肝炎病毒核心抗原及Flt3配体双表达核酸疫苗的构建与体外表达

目的:构建可同时表达乙型肝炎病毒核心抗原(HBcAg)和Flt3配体(FL)胞外段的双表达核酸疫苗。方法:将HBcAg和FL胞外段的基因用PCR方法扩增并分别引入相应的限制性内切酶酶切位点,然后克隆入真核双表达载体pIRES,获得含双基因片段的表达质粒;再取该表达质粒中的内部核糖体切入位点(IRES)序列和基因片段克隆入pJW4303载体中,获得相应的双表达核酸疫苗,经筛选鉴定后,以293T细胞瞬时表达检测两基因的表达水平。结果:构建成功以pIRES、pJW4303为载体的4种HBcAg和FL胞外段的双表达核酸疫苗,体外表达证实HBcAg和FL胞外段均能高效表达,基因位于IRES上游时表达水平较高。结论:用IRES元件实现了HBcAg和FL胞外段双表达核酸疫苗的构建,pJW4303为载体的核酸疫苗两基因表达水平优于pIRES为载体的核酸疫苗。

Construction and in vitro expression of bicistronic DNA vaccines containing hepatitis B virus core antigen and Flt3 ligand genes

Objective:To construct and express bicistronic DNA vaccines containing hepatitis B virus core antigen(HBcAg) and extracellular fragment of Flt3 ligand(FL) genes in vitro. Methods:Cloning sites were introduced to HBcAg and FL genes using polymerase chain reaction(PCR). Two genes were cloned to upstream and downstream internal ribosome entry site(IRES) sequences of pIRES, a bicistronic vector, to construct bicistronic plasmids. Then IRES and gene fragments from digested bicistronic plasmids were cloned to pJW4303, another vector, to construct bicistronic DNA vaccines. After screening and identifying destination DNA vaccines, 293T cells were transfected in order to examine HBcAg and FL genes expression levels. Results:Bicistronic DNA vaccines were constructed successfully and expressed both HBcAg and extracellular FL fragment in vitro and expression level of gene located upstream IRES was higher than downstream. Conclusion:Using IRES element to construct bicistronic DNA vaccine is feasible, and expression of genes in DNA vaccines derived from pJW4303 vector is more superiority than pIRES.