重组BC047440蛋白的表达及其对人肝癌细胞系HepG2增殖的影响

Expression of recombinant BC047440 protein and its effects on proliferation of HepG2 cells in vitro

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 the prokaryotic expression vector of BC047440 gene and to observe the effect of different concentrations of this recombinant protein on proliferation of human HepG2 cells.Methods Total RNA was extracted from HepG2 cells.BC047440 gene fragments were amplified from the total RNA by RT-PCR.The PCR product was cloned into pMD19-T vector and sequenced.The confirmed BC047440 cDNA was cloned into plasmid pET-28a（＋） and transformed into E.coli BL21 where expression of BC047440 proteins was induced by IPTG.The expression product of BC047440 protein was identified by SDSPAGE.Recombinant BC047440 protein was extracted and its effect on proliferation of HepG2 cells was detected by MTT assay and cytometry,respectively.Effect of recombinant BC047440 protein on expression of Ki67 protein in HepG2 cells was detected by Western blotting.Results The prokaryotic expression vector was constructed.The BC047440 gene was cloned,identified,and purified.The proliferation rate of HepG2 cells was 124.0%,136.1%,and 126.6% respectively 48 h after BC047440 protein was transformed into HepG2 cells at the concentration of 1.0,10.0,and 20.0 μg/ml respectively（P 0.05）.Cytometry showed that the proliferation constant of HepG2 cells was 26.1 in control group and 48.6 in experimental group（P 0.05）.The relative expression of Ki67 protein was 0.317 ± 0.062 in experimental group and 0.177 ± 0.037 in control group（P 0.05）.Conclusion Recombinant BC047440 protein can directly promote the proliferation and expression of Ki67 protein and may be a new protein that can stimulate the proliferation of HCC cells.