斑马鱼干扰素γ的克隆表达研究  

Cloning and expression of interferon γ of zebrafish in E. coli BL21(DE3) Expression system

作　　者：高昌[1,2] 齐晓伟[1,2] 穆苑[1,2] 张长勇[2] 祝梅香[2] 王天奇[2] 付淑霞[2] 孙德明[2] 

机构地区：[1]北京协和医学院研究生院,北京100730 [2]国家人口计生委科学技术研究所实验动物中心,北京100081

出　　处：《现代生物医学进展》2009年第13期2421-2423,共3页Progress in Modern Biomedicine

基　　金：北京市应用基础研究与战略高技术课题(20092020)

摘　　要：目的:通过人工诱导斑马鱼干扰素的产生,证实两种斑马鱼干扰素γ基因ifng1-1和ifng1-2的存在,并克隆这两种干扰素基因,构建高效表达载体并做原核表达。方法:通过conA药浴,诱导斑马鱼干扰素γ的表达,即刻提取组织总RNA,并通过RT-PCR方法扩增两种干扰素γ基因。将目的基因连入表达载体pET24a,构建重组载体pET24a-ifng1-1和pET24a-ifng1-2。将载体转化BL21,并用IPTG诱导融合蛋白的表达。产物经SDS-PAGE检测,分析蛋白表达情况。结果:成功克隆了两种斑马鱼干扰素γ基因,构建了pET24a-ifng1-1和pET24a-ifng1-2重组载体,并实现了原核融合表达。结论:两种干扰素γ基因都能够被conA所诱生,且具有相似特性,表明两种干扰素γ都不是假基因,都有可能在免疫系统中发挥作用,本实验为进一步研究两种干扰素γ的功能奠定了基础。Objective： To identify the existence of interferon gamma gene of zebrafish by cloning and constructing the recombinant vectors containing ifngl-1 and ifngl-2 and investigate their expression. Methods： Induce the expression of interferon gamma gene of zebrafish by medicated bath of conA. Extract the RNA immediately and amplify the interferon gamma gene by RT-PCR method after medicated bath, then insert the target gene into the vector of Pet24a and construct the recombinant vector： pET24a-ifngl-1and pET24a-ifiag1-2, then induce the expression offusion protein with IPTG after transforming the vectors into BL21 （DE3）. At last we analyzed the expression of fusion protein after SDS-PAGE electrophoresis. Results： We have cloned the interferon gamma gene of zebrafish： ifngl-1 and ifiagl-2 and constructed the recombinant vectors of pET24a-ifiagl-1 and pET24a-ithgl-2. And finally we completed the expression of pronuclei fussion successfully. Conclusions： The interferon gamma genes： ifiagl-1 and otiagl-2 can be induced by conA similarly.It is suggested that the interferon gamma genes are not pseudogene and both of them may have some effects in the immune system, which lays the foundation for further research of interferon gamma of zebrafish.

关 键 词：斑马鱼 干扰素Γ 原核表达 

分 类 号：Q78[生物学—分子生物学]