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 22×10~3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 3.0 IU/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 study the efficient expression, purification and identification of human growth hormone (hGH) gene in prokaryotes. Methods Double-digested site primers were designed. The hGH cDNA fragment was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into pUC19 plasmid for DNA sequencing. HGH cDNA fragment was cloned into prokaryotic expression vector pBV220 for expression. The purified product was purified by 7mol / L guanidine hydrochloride, denatured, refolded and chromatographed. The purified product was analyzed by SDS-PAGE, high performance liquid chromatography (HPLC) and its activity. The purified product was identified by N-terminal amino acid sequence Assay to identify the expression product. Results The size of the fragment amplified by RT-PCR was consistent with the expected value. The expression product of pBV220-hGH was analyzed by SDS-PAGE. The molecular weight of 22.2 × 10-3 was consistent with the expected result. rhGH expression accounted for 40% of the total bacterial protein. The purity of the expressed product was over 97%, and the specific activity was over 3.0 IU / mg. The purified product was verified by N-terminal 15 amino acid assay, which is recombinant human growth hormone. Conclusion The prokaryotic expression vector pBV220-hGH was successfully constructed and expressed efficiently in E. coli. The purified rhGH with the purity of ≥97% was obtained. The specific activity was over 3.0 IU / mg. This study laid the foundation for the pilot-scale production of rhGH.