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目的克隆香菇C_(91-3)转录本中Unigene1245基因并进行生物信息学分析,研究其编码蛋白的抗肿瘤活性。方法提取香菇C_(91-3)菌丝体总RNA,反转录合成cDNA,经RACE技术获得Unigene1245基因编码序列,通过NCBI对其进行生物信息学分析。设计特异性引物进行PCR扩增,将其克隆到载体pET-32a(+)上,将重组表达质粒热转化至E.coli Rosetta-gami(DE3)中,进行目的蛋白的诱导表达、纯化及复性,通过CCK-8法检测其抗肿瘤活性。结果香菇C_(91-3)转录本Unigene1245基因为YjgF/YER057c/UK114家族成员,成功诱导表达其编码蛋白,CCK-8法结果显示该蛋白对人肝癌HepG2细胞的增殖具有一定抑制作用,且具有一定的时间及浓度依赖性。结论成功克隆并诱导表达Unigene1245基因,并鉴定其具有抑制HepG2细胞增殖的能力,为后续探究抗肿瘤机制奠定了基础。
Objective To clone the gene Unigene1245 of C_ (91-3) transcript of lentinus edodes and analyze its bioinformatics to study its antitumor activity. Methods The total RNA of mycelia of C_ (91-3) was extracted and cDNA was reverse transcribed. The coding sequence of Unigene1245 was obtained by RACE and bioinformatics analysis was carried out by NCBI. The specific primers were designed for PCR amplification, cloned into the vector pET-32a (+), and the recombinant expression plasmids were heat-transformed into E.coli Rosetta-gami (DE3) to induce expression, purification and complexation of the target protein The anti-tumor activity was detected by CCK-8 assay. Results The Unigene 1245 gene of M_ (91-3) transcript of Lentinula edodes was a member of YjgF / YER057c / UK114 family and was successfully induced to express its encoded protein. The results of CCK-8 assay showed that this gene could inhibit the proliferation of HepG2 cells, A certain time and concentration-dependent. Conclusion The gene of Unigene1245 was successfully cloned and induced, and its ability to inhibit the proliferation of HepG2 cells was identified, which laid the foundation for further exploration of antitumor mechanisms.