香菇转录本Unigene 10627过氧化物酶结构域的克隆表达及抗氧化活性研究

Cloning,expression and antioxidant activity of Unigene 10627 from the transcript of Lentinula edodes C_(91-3)

目的分析香菇C91-3转录本Unigene 10627基因的结构域,并对其进行诱导表达,初步探讨所得目的蛋白的抗氧化活性。方法将香菇C91-3菌丝体中总RNA提取出来,通过反转录获得cDNA文库,根据反转录结果,通过3′-RACE(Rapid Amplification of cDNA Ends),5′-RACE技术扩增获取基因全长。通过生物信息学方法预测基因的结构域,利用PCR技术扩增该目的片段,将扩增产物连接到原核表达载体pET32a(+)上,采用热转化法转至E.coli Rosetta-gami(DE3)中进行诱导表达,对目的蛋白进行分离、纯化及鉴定。采用二苯代苦味酰自由基(2,2-diphenyl-1-picrylhydrazyl,DPPH)法检测蛋白的抗氧化活性。结果成功获得基因全长,预测结果表明该基因具有染料脱色过氧化物酶结构域(DyP_Perox),双酶切结果证实目的片段成功插入,并通过诱导表达获得目的蛋白。结论目的蛋白成功表达,且具有抗氧化活性,为进一步研究其生物学活性提供基础。

Objective To analyse the structure domain of the gene transcript Unigene 10627 in Lentinula edodes C91-3by using bioinformatic techniques,and evaluate the antioxidant capacity of the harvested proteins.Methods The total RNAs were extracted from the mycelia of Lentinula edodes C91-3,and cDNA was synthesized by reverse transcription.Based on the transcriptome sequence,the primers were designed and the full-length gene was produced by using the 3′-Full RACE and 5′-Full RACE methods.The functional domains of Unigene 10627 was predicted through the NCBI Database online.The structure domain was amplified by PCR with the specific primers.The cloned products were connected to pET-32a（＋）vector,then transformed to E.coli Rosetta-gami（DE3）.The recombinant proteins were harvested by inducing,purifying and refolding.The ability of the proteins to scavenge DPPH（2,2-diphenyl-1-picrylhydrazyl）free radical was evaluated.Results The full-length gene was harvested successfully.The predict results confirmed that the structure domain was DyP_Perox.The recombinant sequence was confirmed by enzyme digestion.Conclusion The recombinant proteins are harvested successfully,and were confirmed to have obvious antiradical capacity.These results provide a compacted foundation for an in-depth study of the proteins.