p21^(Ha-ras)原核表达载体的构建、表达及纯化的研究

Construction of prokaryotic expression plasmid PET-28a(+)-Ha-Ras and expression and purification of p21ras

目的:构建PET-28a(+)-Ha-ras原核表达质粒,并表达、纯化得到p21ras蛋白,为研究p21ras在肿瘤发病中的作用和机制以及p21ras细胞内抗体抗肿瘤的研究奠定基础。方法:培养人肝癌细胞株7703,提取总RNA,通过RT-PCR特异扩增出Ha-ras cDNA,然后应用TA克隆策略将纯化后的Ha-ras插入至中间载体pMD18-T vector中得到重组质粒PMD-18T vector-ras,重组质粒PMD-18Tv ector-Ha-ras经过BamHⅠ和HindⅢ双酶切后纯化回收得到具有黏性末端的Ha-ras cDNA,将PET-28a(+)同样经过BamHⅠ和HindⅢ双酶切后纯化回收得到与ras cDNA相同的黏性末端的线形质粒片段,将具有黏性末端的ras cDNA与酶切后的PET-28a(+)定向连接,构建出重组质粒PET-28a(+)-Ha-ras,经酶切鉴定,并通过核苷酸序列测序证实。将鉴定正确的PET-28a(+)-Ha-ras转入感受态BL-21(DE3)中诱导表达,利用组氨酸"标签"(His-Tag)对p21ras进行金属螯合亲和层析纯化。结果:测序分析证实,克隆入PET-28a(+)的Ha-ras序列与Genbank登录号为"NM_005343"的Ha-ras cDNA序列一致,将重组质粒PET-28a(+)-Ha-ras转化BL21(DE3),用IPTG诱导目的蛋白表达。SDS-PAG和蛋白纯化结果表明,PET-28a(+)-Ha-Ras在E.coli中获得可溶性高表达,经Ni-NTA-树脂柱亲和层析纯化得到了PAGE纯的p21ras蛋白。结论:成功构建了原核表达载体PET-28a(+)-Ha-ras,并经表达、纯化得到活性形式的p21ras蛋白,从而为研究p21ras在肿瘤发病中的作用和机制以及P21ras细胞内抗体抗肿瘤的研究奠定了基础。

Objective To construct the prokaryotic expression plasmid PET-28a（＋）-Ha-ras and to obtain purified protein of p21ras. Methods Using RT-PCR, Ha-ras cDNA was generated from amplification of the total RNA extracted from human hepatocellular carcinoma cell line 7703. The purified product of Ha-ras cDNA was then inserted into the pMD18-T vector to form a recombinant pMD18-T vector-Ha-ras. The recombinant plasmid was cut by restriction enzymes BamH/Hind and then purified to become Ha-ras cDNA with viscous ends, as was PET-28a（＋）to become a linear plasmid fragment with the same viscous ends as Ha-ras cDNA. The Ha-ras cDNA was combined with the PET-28a （＋） that had been cut by the enzymes to construct the recombinant plasmid PET-28a（＋）-Ha-ras. This plasmid was identified via restriction enzymes and was finally confirmed by the sequencing of the nucleotides. The correct PET-28a （＋）-Ha-ras was transformed into BL21 （DE3） to induce expression. The p21ras was purified by immobilized metal ion affinity chromatography with a histidine ＂label＂ （His-Tag）. Results Sequence analysis showed that the Ha-ras cDNA sequence of PET-28a（＋）-Ha-Ras had the identical sequence as the Ha-ras cDNA sequence printed in GenBank （accession NO.NM - 005343）. SDS-PAGE and Western-blot confirmed PET-28a（＋）-Ha-ras was highly expressed in E.coli and the purified p21ras protein was generated by using Ni-NTA affinity chromatography. Conclusions The prokaryotic expression plasmid PET-28a（＋）-Ha-ras has been constructed successfully. The successful production of active p21ras protein lays, the foundation for exploring the effect of p21ras on the genesis of neoplasms and for the research on the intracellular antibody of p21ras against neoplasms.