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目的通过生物信息学方法分析Latcripin-8的基因序列、功能域片段,构建重组质粒,表达含Pkinase结构域基因的目的蛋白并研究重组蛋白的抗肿瘤活性。方法从香菇C91-3菌丝体中提取总RNA,经反转录成cDNA文库。高通量测序获得转录组mRNA,经对比获得目的序列后以RACE技术扩增。经Pfam数据库比对可知其具有Pkinase结构域。设计引物,PCR扩增并与原核表达载体pET32a(+)进行连接,转至E.coli Rosetta-gami(DE3)中进行蛋白诱导表达,经纯化、复性后获得目的蛋白并鉴定。CCK8法检测其对人肺腺癌A549细胞、人胃癌SGC-7901细胞、人肝癌HepG2细胞及人乳腺癌MCF-7细胞的增殖抑制率。结果蛋白诱导成功且该蛋白对不同种类的肿瘤细胞抑制效果不同,其中对人胃癌SGC-7901细胞的增殖抑制程度最为明显,并表现出时间和浓度依赖性。结论经Latcripin-8基因诱导表达出的蛋白具有抗肿瘤功效,为进一步深入研究抗肿瘤方面的活性提供了初步条件,有助于肿瘤治疗作用的研究。
OBJECTIVE: To analyze the gene sequence and functional domain of Latcripin-8 by bioinformatics methods, construct the recombinant plasmid, express the target protein containing Pkinase domain gene and study the anti-tumor activity of the recombinant protein. Methods Total RNA was extracted from mycelium of Mushroom Mushroom C91-3 and reverse transcribed into cDNA library. Transcriptome mRNA was obtained by high-throughput sequencing. The target sequence was obtained by comparison and amplified by RACE. The Pfam database shows that it has a Pkinase domain. The primers were designed, amplified by PCR and ligated with the prokaryotic expression vector pET32a (+). The recombinant plasmid was transformed into E.coli Rosetta-gami (DE3) for protein induction. After purification, renaturation and identification of the target protein. CCK8 assay was used to detect the proliferation inhibition rate of human lung adenocarcinoma A549 cells, human gastric cancer SGC-7901 cells, human hepatoma HepG2 cells and human breast cancer MCF-7 cells. Results The protein was successfully induced and the inhibitory effect of this protein on different types of tumor cells was different. The inhibition of SGC-7901 cells proliferation was most obvious and showed time and concentration dependence. Conclusion The protein induced by Latcripin-8 gene has the anti-tumor effect, which provides initial conditions for further study on the anti-tumor activity and is helpful for the study of tumor therapeutic effect.