摘要
构建人髓样分化蛋白 2 (humanmyeloiddifferentiatonprotein 2 ,hMD 2 )与谷胱甘肽巯基转移酶 ( glutathione S transferase,GST)的融合蛋白(hMD 2 /GST)表达载体PGEX 4T 1/hMD 2 ,在大肠杆菌中融合表达hMD 2 /GST。以真核表达载体PEF BOS/hMD 2为模板 ,用PCR法在MD 2编码序列上下游引入酶切位点和终止密码子 ;将hMD 2编码序列克隆入原核表达载体PGEX 4T 1的相应酶切位点 ;用PCR和酶切筛选、鉴定重组质粒PGEX 4T 1/hMD 2 ,并对插入基因片断测序 ;重组质粒PGEX 4T 1/hMD 2转化大肠杆菌 ,经IPTG诱导后用SDS PAGE分析表达产物。结果PCR、酶切鉴定和序列测定证实MD 2编码序列正向插入原核表达载体PGEX 4T 1的相应酶切位点 ,片断与PCR扩增产物大小相同、序列无误、阅读框架正确 ;SDS PAGE证实hMD 2 /GST在大肠杆菌中表达 ,表达量约占菌体总蛋白的 3 0 %。说明成功构建了PGEX 4T 1/hMD 2载体 ,hMD 2 /GST融合蛋白在大肠杆菌中得到初步表达 ,为MD
To design and construct an expression vector,PGEX-4T-1/MD-2, and to express human myeloid differentiation protein-2(hMD-2) and glutathione-S-transferase (GST) fusion protein in E. coli, the EcoRI/SalI sites and stop code were incorporated into the hMD-2 encoding fragment by PCR. After digesting with EcoRI/SalI, the hMD-2 encoding fragment was cloned into the expression vector PGEX-4T-1 at the corresponding sites. The positive clones selected with PCR and restriction endonuclease digestion were sequenced and the expression of GST/hMD-2 fusion protein in E. coli BL21 was analyzed with SDS-PAGE after induced by 0.4mmol/L IPTG for 3 to 5 hours. The hMD-2 encoding fragment containing stop code was correctly inserted into the expression vector PGEX-4T-1 and this was confirmed by PCR, double-enzyme identification and sequencing. The SDS-PAGE analysis indicated that the GST/hMD-2 fusion protein was successfully expressed in E. coli and the yield of the fusion protein was 30 percent of bacterial total protein. The construction of the expression vector PGEX-4T-1/hMD-2 and the expression of fusion protein GST/hMD-2 in E. coli would be useful for further investigation of MD-2.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2003年第3期206-208,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家重点基础研究发展规划项目资助课题 (编号G1 9990 542 0 3)
关键词
重组融合蛋白类
谷胱甘肽巯基转移酶
人髓样分化蛋白-2
遗传载体
recombinant fusion proteins
glutathione-S-transferase
human myeloid differentiaton protein-2
genetic vectors
