汉坦病毒H8205株G1-人源IL-2融合基因的克隆与表达  

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

作　　者：孙辉[1] 关武祥[1] 黄汉菊[1] 

机构地区：[1]华中科技大学同济医学院基础医学院病原生物学系,武汉430030

出　　处：《华中科技大学学报（医学版）》2003年第6期578-581,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No 3 0 170 819)

摘　　要：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 B IL 2。同样采用PCR扩增汉坦病毒H82 0 5株G1基因片段 ,将回收的G1片段经双酶切插入到pcD NA3 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一分子量为 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 。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.

关 键 词：汉坦病毒 H8205株 融合基因 克隆 表达 糖蛋白G1 白细胞介素2 

分 类 号：R373[医药卫生—病原生物学]