人脂联素球型结构域原核表达重组质粒的构建及体外表达

Construction and expression of a prokaryotic vector of recombinant human adiponectin global domain

目的通过分子克隆表达人脂联素基因球型结构域(gAd),为进一步研究提供实验基础。方法应用RT-PCR方法从人大网膜脂肪组织总RNA中扩增出gAd cDNA,克隆入载体pMD18-T、pET32a(+)中,形成重组质粒gAd-pET32a(+)。筛选出阳性克隆,通过限制性内切酶酶切、PCR和测序进行鉴定,并在体外进行小量表达。结果从脂肪组织总RNA中扩增得到412 bp片段gAd基因,其cDNA序列与GenBank人gAd基因序列相同,并在体外表达得到相对分子质量为34 000的重组蛋白。结论成功构建了gAd基因原核表达质粒并实现体外的小量表达。

Objective To construct and express the recombinant human adiponectin （gAd） global domain. Methods gAd complementaryDNA （cDNA） wasobtainedfi-omhuman fat tissuebyRT-PCR. The PCRproduct was clonedinto the vectorpMD 18-T a nd the prokaryotic expression vector pET32a （＋）. The recombinant vector was identified by digestion with double restriction endonucleases SalI and EcoRl, PCR and sequence analysis. The recombinant plasmid containing gAd gene was transformed into E. coli BL21 （DE3）, and the expression of the fusion protein His-gAd was induced by IPTG. Results The gAd cDNA of 412 bp was obtained fi-om the total RNA of the fat tissue and verified by sequence analysis. Conclusion The recombinant plasmid could stably express the 34-kD fusion protein His-gAd in the engineered bacteria in the form of inclusion bodies.