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目的构建原核表达载体,制备重组BC047440蛋白并观察不同浓度的重组蛋白对HepG2细胞增殖的影响。方法提取肝癌组织总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增BC047440cDNA,克隆入质粒pMD19-T,转化E.coliDH5α,酶切目的基因片段,插入质粒pET-28a(+),转化E.coliBL21,IPTG诱导蛋白表达,SDS-PAGE电泳鉴定表达产物,提取重组BC047440蛋白,MTT法和流式细胞术检测不同浓度重组蛋白对HepG2细胞增殖的影响,Westernblot检测重组蛋白作用后HepG2细胞表达Ki67蛋白的变化。结果克隆、鉴定BC047440基因,构建其原核表达载体,成功制备并纯化BC047440重组蛋白。1.0、10.0、20.0μg/ml浓度重组BC047440蛋白分别作用HepG2细胞48h后,细胞增殖率分别为124.0%、136.1%、126.6%,与对照组相比,差异有统计学意义(P<0.05)。流式细胞术结果提示,对照组的细胞增殖常数(PI)为26.1,重组蛋白处理组PI为48.6,二者差异具有统计学意义(P<0.05)。重组BC047440蛋白作用HepG2细胞后,Ki67蛋白的相对表达量为(0.317±0.062),对照组的相对表达量为(0.177±0.037),二者差异具有统计学意义(P<0.05)。结论制备的重组BC047440蛋白具有直接促进HepG2细胞增殖和促进其表达Ki67蛋白的作用,可能是一种新的促进肝癌增殖蛋白。
Objective To construct prokaryotic expression vector and prepare recombinant BC047440 protein and observe the effect of different concentrations of recombinant protein on the proliferation of HepG2 cells. Methods Total RNA was extracted from hepatocellular carcinoma tissues. BC047440 cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR), cloned into plasmid pMD19-T and transformed into E.coli DH5α. The target gene fragment was digested and inserted into plasmid pET-28a ) Were transformed into E.coliBL21 and induced by IPTG. The expression product was identified by SDS-PAGE electrophoresis. The recombinant BC047440 protein was extracted and the effects of different concentrations of recombinant protein on the proliferation of HepG2 cells were detected by MTT assay and flow cytometry. HepG2 cells express Ki67 protein. Results The BC047440 gene was cloned and identified. The prokaryotic expression vector was constructed and the BC047440 recombinant protein was successfully prepared and purified. The proliferation rates of HepG2 cells treated with 1.0, 10.0 and 20.0μg / ml recombinant BC047440 protein were 124.0%, 136.1% and 126.6%, respectively. The difference was significant compared with the control group (P <0.05). Flow cytometry results showed that the cell proliferation constant (PI) of the control group was 26.1, and the PI of the recombinant protein treatment group was 48.6, the difference was statistically significant (P <0.05). The relative expression of Ki67 protein in HepG2 cells treated with recombinant BC047440 protein was (0.317 ± 0.062), compared with that of control group (0.177 ± 0.037), the difference was statistically significant (P <0.05). Conclusion The recombinant BC047440 protein can directly promote the proliferation of HepG2 cells and promote the expression of Ki67 protein, which may be a new protein that promotes proliferation of HCC.