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目的通过对香菇C91-3转录组进行筛选,并克隆表达含RCC1(染色体浓缩调控蛋白)和ANK(锚蛋白)结构域的Unigene8290基因,研究其抗肿瘤活性。方法从香菇C91-3菌丝体中提取总RNA,反转录合成cDNA,并利用Rapid Amplification of cDNA Ends(RACE)技术获得基因全长。生物信息学分析其结构域。设计特有引物,采用PCR技术扩增该结构域,将其克隆产物与pET-32a(+)载体连接,热转化至E.coil Rosetta-gami(DE3)中诱导表达,纯化、复性后,通过MTT法研究其抗肿瘤活性。结果生物信息学显示Unigene8290基因具有RCC1和ANK结构域,琼脂糖凝胶电泳与测序结果显示Unigene8290基因的结构域片段与pET-32a(+)载体重组成功,SDS-PAGE与Western blot结果显示Unigene8290蛋白成功表达,MTT结果显示该重组融合蛋白对HepG2细胞生长具有明显的抑制作用,并且其抑制作用存在一定的时间和浓度依赖性。结论成功诱导Unigene8290的RCC1和ANK结构域蛋白表达,并初步鉴定其具有抑制HepG2肿瘤细胞增殖活性的功能。
Objective To investigate the antitumor activity of Unigene8290 gene by screening the C91-3 transcriptome of Mushroom and cloning the Unigene8290 gene, which contains RCC1 (chromosomal condensation regulatory protein) and ANK (ankyrin) domain. Methods The total RNA was extracted from the mycelium of Mushroom C91-3 and the cDNA was reverse transcribed. The full-length cDNA was obtained by Rapid Amplification of cDNA Ends (RACE). Bioinformatics analysis of its domains. Specific primers were designed and the domain was amplified by PCR. The cloned product was ligated into pET-32a (+) vector and induced to heat by E.coil Rosetta-gami (DE3). After purification and refolding, MTT assay of its anti-tumor activity. Results Bioinformatics showed that the Unigene8290 gene had RCC1 and ANK domains. The results of agarose gel electrophoresis and sequencing showed that the fragment of Unigene8290 was successfully recombined with pET-32a (+) vector. SDS-PAGE and Western blot showed that the Unigene8290 protein The results of MTT showed that the recombinant fusion protein had a significant inhibitory effect on the growth of HepG2 cells, and its inhibitory effect was dependent on time and concentration. Conclusion The expression of RCC1 and ANK domains of Unigene8290 was successfully induced and its function of inhibiting the proliferation activity of HepG2 tumor cells was preliminarily identified.