IL-2/Fc融合基因真核表达载体的构建和表达

Construction and expression of eukaryotic expression vector bearing fusion gene of human IL-2 cDNA gene and Fc gene

目的 :构建含人IL 2cDNA基因和免疫球蛋白Fc片段融合基因的真核表达载体pcDNA 3.1IL 2 /Fc ,并在真核细胞中表达 ,以期用于乙型肝炎病毒 (HBV)DNA疫苗的研究。 方法 :应用重叠延伸剪切技术 (SOE)经两次PCR获得嵌合基因片段IL 2 /Fc,回收后克隆到中间 pGEM TEasyTA克隆载体 ,得到合适的酶切位点后 ,用双粘端连接法转克隆入真核表达载体pcDNA 3.1中 ,得到真核重组载体 pcDNA 3.1IL 2 /Fc。然后用脂质体法转染SP2 / 0细胞。 结果 :对重组载体进行限制性酶切鉴定及测序分析 ,证明连接正确 ;经ELISA检测证实 ,该重组载体能够在真核细胞中外分泌表达插入的外源性基因编码的融合蛋白。 结论 :成功构建了真核表达载体 pcDNA 3.1IL 2 /Fc ,并在SP2 / 0细胞中有效表达。

Objective:To construct and express a recombinant eukaryotic expression vector bearing fusion gene of human IL-2 cDNA gene and Fc fragment. Methods:Technique of splicing by overlapping extension and two times PCR were used, and fusion gene fragment was obtained and cloned into pGEM-T Easy TA cloning vector to get suited enzymed sites. Recombinant eukaryotic expression vector pcDNA 3.1 IL-2/Fc was constructed by double adhesive terminal ligation. Then the recombinant vector was transferred into SP 2/0 cells using Lipofectamine. Results:The recombinant vector was identified by digestion with restriction enzymes and confirmed by DNA sequencing analysis. And exocrine expression of the fusion gene in eukaryotic cells was detected by ELASA method. Conclusion:The relative efficient expression of the fusion gene in SP 2/0 cells might provide an experimental basis for improving the efficiency of DNA vaccine.