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目的:构建人类核糖核酸酶9(human ribonuc lease9,hRNase9)的重组表达载体。方法:从成年人附睾组织提取了总RNA,通过RT-PCR扩增了hRNase9编码成熟肽的基因片段,将其克隆入载体pET25b(+)的NdeI和EcoRI位点之间,构建了重组表达载体pET25b(+)-hRNase9,并通过PCR法、限制性内切酶消化法和DNA序列测定加以证实,以IPTG诱导重组载体转化宿主菌E.coliBL21(DE3)表达重组hRNase9蛋白。结果:重组载体pET25b(+)-hRNase9构建成功,SDS-PAGE显示重组载体转化宿主菌E.coliBL21(DE3)表达了Mr约为21000的重组hRNase9蛋白。结论:成功的扩增了hRNase9编码成熟肽的基因片段,构建了重组表达载体pET25b(+)-hRNase9,为下一步研究奠定了基础。
Objective: To construct a recombinant expression vector of human ribonuc lease9 (hRNase9). METHODS: Total RNA was extracted from adult epididymis tissue. The gene fragment encoding mature peptide of hRNase9 was amplified by RT-PCR and cloned into the NdeI and EcoRI sites of vector pET25b (+) to construct a recombinant expression vector pET25b (+) - hRNase9. The recombinant plasmid pET25b (+) - hRNase9 was confirmed by PCR, restriction endonuclease digestion and DNA sequencing. The recombinant hRNase9 protein was expressed in E. coli BL21 (DE3) transformed by IPTG. Results: The recombinant vector pET25b (+) - hRNase9 was constructed successfully. The recombinant hRNase9 was expressed by SDS-PAGE. The recombinant hRNase9 with Mr of 21000 was expressed in E. coli BL21 (DE3). Conclusion: The gene fragment encoding mature peptide of hRNase9 was successfully amplified and the recombinant expression vector pET25b (+) - hRNase9 was constructed, which laid the foundation for further study.