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目的 :构建干扰素 α- 8(IFNα- 8)与乙型肝炎 (HB)病毒 (HBV) - S2 S融合蛋白的真核表达质粒 p VAX1/IFNα- 8- S2 S,作为一种高效能 HB DNA疫苗的备选对象。方法 :以特定引物分别从正常胎肝组织及 HB患者血清中扩增出 IFNα- 8与 HBV- S2 S基因片段 ,克隆至相应载体。再以连接引物扩增出 IFNα- 8与 HBV- S2 S融合基因片段 ,并将该基因片段克隆至真核细胞表达载体 p VAX1,然后扩增目的基因测序 ,确认无误后 ,转染 SP2 / 0细胞 ,并分别检测目的基因 m RNA,了解其短暂表达与稳定表达情况。结果 :所构建的质粒经过酶切电泳后证实分子量与预期相符 ,目的片断 IFNα- 8- S2 S经测序证实 IFNα- 8序列与 Genebank公布的序列相符。HBV- S2 S存在 3处三联密码子的无义突变。 2处有义突变是 70 3- 70 5位 CCG突变为 CAG,799～ 80 1位 ATA突变为 ATG。表达质粒转染 SP2 / 0细胞后 ,均检测到短暂表达及稳定表达之目的基因 m RNA。结论 :经分析表明 ,HBV- S2 S片段存在的两处有义突变并不影响该蛋白的空间构象及关键抗原决定簇的性质。可以认为本研究完成了真核表达质粒 p VAX1/ IFNα- 8- S2 S的构建 ,并成功检测到目的基因的表达 ,为制造高效能 HB DNA疫苗提供了新的备选对象。
OBJECTIVE: To construct eukaryotic expression plasmid p VAX1 / IFNα-8- S2 S of interferon α-8 (IFNα-8) and hepatitis B virus (HBV)-S2 S fusion protein as a high-potency HB Alternative to DNA vaccine. Methods: IFNα-8 and HBV-S2 S gene fragments were amplified from the normal fetal liver tissue and HB serum by specific primers and cloned into the corresponding vector. Then, the fusion gene of IFNα-8 and HBV-S2 S was amplified by ligated primer, and cloned into eukaryotic expression vector p VAX1. Then the target gene was amplified and sequenced. Cells, and respectively detect the target gene m RNA to understand its transient expression and stable expression. Results: The constructed plasmids were verified by restriction endonuclease electrophoresis. The target fragment of IFNα-8- S2 S was confirmed by sequencing. The sequence of IFNα-8 was in accordance with that of Genebank. There are three nonsense mutations in HBV-S2 S triplet codons. The two sense mutations were 70-370-5 CCG to CAG, and the 799-801 ATA was mutated to ATG. After transfection of SP2 / 0 cells with expression plasmids, both the transiently expressed and stably expressed m RNA were detected. CONCLUSION: The analysis shows that the two sense mutations in HBV-S2 S fragment do not affect the spatial conformation and the key antigenic determinants of the protein. It is considered that the eukaryotic expression plasmid p VAX1 / IFNα- 8- S2 S was constructed and the expression of the target gene was successfully detected, thus providing a new candidate for manufacturing high-performance HB DNA vaccines.