HBcAg基因和PreS1(1～65)肽基因真核重组载体的构建和鉴定

Construction and identification of the eukaryotic recombination vector of HBcAg and PreS1(1～65)

目的：构建并鉴定包含人乙型肝炎病毒（HBV）HBcAg基因和PreS1（1～65）肽基因的真核重组载体。方法：采用PCR法扩增HBcAg基因，并通过基因突变在79～80位氨基酸处生成一个NheⅠ酶切位点，然后将这段基因连接到真核表达载体pIRES2-EGFP启动子下游的EcoRⅠ和BamHⅠ之间。同时PCR扩增PreS1第1～65氨基酸基因，并在其两端分别引入NheⅠ酶切位点，通过酶切、连接，将这段基因插入到HBcAg基因的NheⅠ酶切位点上。将构建的重组载体pIREScS1转化大肠杆菌DH5α，分别用上述内切酶双酶切及DNA测序鉴定重组质粒。结果：酶切鉴定示，插入片段大小均与预计相符。测序结果与已知序列结果一致。结论：成功构建了HBcAg基因和PreS1（1～65）肽基因的真核重组载体。

Objective： To construct and identify the eukaryotic recombination vector including of HBcAg and PreS1 （ 1 - 65 ） . Methods： The core antigen gene of HBV was amplified by PCR. A MCS of Nhe Ⅰ was created in the 79 - 80AA of HBcAg by gene mutation. This gene was directly inset.ted into the downstream （ between the MCS of EcoR Ⅰ and BamH Ⅰ） of CMV promoter in the plRES2-EGFP. Before, the gene of PreS1 （ 1 - 65） was amplified by PCR, and two MCSs of Nhe Ⅰ was created in the both sides of PreS1 （ 1 - 65） gene., respectively. Then, the PreS1 （ 1 - 65 ） gene was directly inset.ted into HBcAg gene. This recombination vector （pIREScS1） was transformed into the DH5and identified by cutting with EcoR Ⅰ and BamH Ⅰ, and DNA sequencing assay. Results： The inserted fragment was consistent with prediction. The outcome of DNA sequencing was consistent with the reported sequences. Conclusion： The eukaryotic recombination vector （pIREScS1） including of HBcAg and PreS1 （ 1 - 65） was constructed successfully.