汉坦病毒H8205株G1-人源IL-2融合基因的克隆与表达

目的 构建融合基因pcDNA3．1／His-B-IL-2-G1，为进一步研究汉坦病毒新型裸DNA疫苗奠定基础。方法 采用PCR从含有人源IL-2的质粒中扩增出IL-2片段，将人源IL-2插入真核表达载体pcDNA3．1／His—B中，构建的质粒命名为pcDNA3．1／His-昏IL-2。同样采用PCR扩增汉坦病毒H8205株G1基因片段，将回收的G1片段经双酶切插入到pcDNA3．1／His-B-IL-2，构建成新的质粒pcDNA3．1／His-B-IL-2-G1。采用脂质体介导包裹，转入NIH3T3细胞中进行瞬时表达；采用原位杂交观察细胞内的基因表达，SDS-PAGE法作重组质粒pcDNA3．1／His-B-IL-2-G1瞬时表达产物的鉴定。结果 融合基因可转录并表达-分子量为78kD左右的蛋白，对照组则未见电泳条带出现。结论 成功构建pcDNA3．1／His-B-IL-2-G1融合基因，融合基因能在真核细胞中瞬时表达。

Cloning and Expression of the Fusion Gene of G1 Segement of Hantavirus H8205 Strain and IL-2 Gene of Human

Objective To clone a recombinant plasmid co nt aining hantavirus G1 gene and IL-2 gene for the purpose of developing a new DNA vaccine against Hantavirus.Methods IL-2 from the plasmid which was donated by the depart ment of molecular biology was amplified by routine PCR. The fragment was inserte d into downstream of the vector pcDNA3 1/His-B to obtain the recombinant eukar yotic expression plasmid pcDNA3 1/His-B-IL-2 identified by the methods of en zyme digestion, routine PCR and sequence analysis. Similarly, the open reading f rame of G1 gene from the plasmid was amplified and the enzyme-digested fragment was ligated to pcDNA3 1/His-B-IL-2 digested with the same enzymes to fom th e recombinant expression plasmid pcDNA3 1/His-B-IL-2-G1. The recombinant pl asmid pcDNA3 1/His-IL-2-G1 was transfected into eukaryotic NIH3T3 cells by l ipofectamine-mediated transfection. mRNA of the recombinant plasmid was detecte d by the method of hybrization in situ with the self-designed probes. SDS- PAGE method was used to identify the transient expression of the recombinant pla smid pcDNA3 1/His-B-IL-2-G1. Results A protein with 78 kD was transcribed and expressed by the fusion gene, but in the control group no electrophoresis strand was found. Conclusion The fusion gene pcDNA3 1/His-IL-2-G1 is cloned s uccessfully and can be transiently expressed in eukaryotic cells.