人MD-2/GST融合蛋白在大肠杆菌的表达

构建人髓样分化蛋白－2（human myeloid differentiation proterin-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30％，说明成功构建了PEGX－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－2后续研究作了必要的准备。

THE EXPRESSION OF HUMAN MD-2/GST FUSION PROTEIN IN E. coli

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.