人生长激素基因的克隆、高效表达及纯化

Cloning, expression and purification of human growth hormone cDNA

目的研究人生长激素(hGH)基因在原核中的高效表达、纯化及鉴定.方法设计带双酶切位点引物,采用逆转录-聚合酶链反应(RT-PCR)方法获得hGH cDNA片段,并亚克隆入pUC19质粒中进行DNA序列测定.将hGH cDNA片段克隆入原核表达载体pBV220中进行表达.表达产物经7mol/L盐酸胍裂解变性、复性、层析等一系列纯化研究,纯化产物经SDS-PAGE电泳,高压液相色谱法(HPLC)纯度分析及活性测定,并通过N末端氨基酸序列测定鉴定表达产物.结果 RT-PCR扩增所得片段大小与预期值一致.pBV220-hGH表达产物经SDS-PAGE电泳显示,分子量为22×103与预计结果相符.rhGH表达量占菌体总蛋白量的40%.表达产物纯化后,纯度达97%以上,比活性大于3.0IU/mg.纯化产物经N末端15个氨基酸测定验证,为重组人生长激素.结论成功构建了pBV220-hGH原核表达质粒,并在大肠杆菌中高效表达,表达产物经纯化得到纯度≥97%的rhGH,比活性大于3.0 IU/mg.该研究为rhGH的中试生产奠定了基础.

Objective To investigate the high--level expression, purification and identification of recombined human growth hormone cDNA in E. coli. Methods With primer containing EcoRI and PstI sites, rhGH cDNA was amplified by RT-PCR and inserted into pUC19 for sequencing. Then rhGH was recombined with expression vector pBV220 and expressed in E. coli. The expression product was purified by ion-exchange chromatography and gel filtration and demonstrated by SDS-PAGE, HPLC profile and i- dentified by N-terminal amino acids sequence analysis. Results Sequence analysis of the amplified frag- ment confirmed that the cloned cDNA was hGH. The expression product was identical with that previous- ly reported by SDS-PAGE. The highest expression level accounted for 40% of the total cellular protein. The purity and specific activity of the purified rhGH reached above 97% and 3. 0 IU/mg respectively. The sequences of 15 amino acids in N-terminal of productwere consistent with hGH. Conclusion The pBV220-hGH prokaryotic expression vector was successfully constructed and highly expressed in E. coli. The purity and specific activity of the purified rhGH reached above 97% and 3. 0 IU/mg respectively.