重组人生长激素的原核分泌表达及其活性分析

Prokaryotic expression and activity of recombinant human growth hormone

目的构建重组人生长激素(recombinant human growth hormone,rhGH)分泌表达质粒,进行原核表达,并分析其活性。方法人工合成包含phoA启动子、STⅡ信号肽序列及hGH基因优化序列的目的基因,与pBR322载体连接,构建重组克隆质粒pBR322-hGH;再将目的基因亚克隆至pACYC184载体,构建重组表达质粒pAC-hGH,进行酶切鉴定及测序分析。将鉴定正确的重组表达质粒pAC-hGH转化至感受态大肠埃希菌W3110中,构建重组工程菌,经诱导表达后进行DEAE-Sepharose FF纯化,纯化产物经SDS-PAGE、分子排阻色谱、N-末端氨基酸序列及活性分析。结果重组表达质粒经酶切及测序鉴定,构建正确。表达产物经SDS-PAGE分析可见相对分子质量约22 000的目的条带,菌体相对表达量达30%以上;纯化产物电泳纯度达95%以上,分子排阻色谱保留时间及N-末端15个氨基酸均与国家标准品一致,比活性大于3.0 IU/mg。结论成功构建了分泌表达rhGH的重组质粒,且获得了纯度及活性均较高的rhGH,为其产品的开发奠定了基础。

Objective To construct the vector for secretory expression of recombinant human growth hormone（rhGH）in prokaryotic cells and analyze the activity of expressed product. Methods The target gene containing phoA promoter, STⅡ signal peptide sequence and optimized hGH gene sequence was artificially synthesized, and inserted into vector pBR322 vector to construct recombinant cloning plasmid pBR322-hGH. Then, the target gene was subcloned to vector pACYC184,and the constructed recombinant plasmid pAC-hGH was identified by restriction analysis and sequencing, and transformed to competent E. coli W3110. The expressed product was purified by DEAE-Sepharose FF chromatography, analyzed by SDS-PAGE, molecular exclusion chromatography and N-terminal amino acid sequencing, and determined for activity.Results Restriction analysis and sequencing proved that recombinant plasmid was constructed correctly. The relative molecular mass and relative expression level of target protein were about 22 000 and more than 30% respectively, while the purity reached more than 95% after purification, the retention time of HPLC was consistent with that of national standard, and the activity was more than 3. 0 IU/mg. Conclusion The recombinant plasmid for secretory expression of rhGH in prokaryotic cells was successfully constructed, and rhGH with high purity and activity was obtained, which laid a foundation of development of rhGH.