C7orf69基因的克隆表达及生物信息学分析

Cloning, expression and bioinformatics analysis of gene C7orf69

目的体外克隆表达并纯化C7orf69重组蛋白,对C7orf69蛋白进行生物信息学分析。方法 PCR扩增C7orf69目的基因片段,构建原核表达载体,转化BL21,IPTG诱导目的蛋白表达,SDS-PAGE和Western blot鉴定,Ni-NTA纯化目的蛋白。结果扩增获得序列完全正确的C7orf69基因片段(自然变异体),重组蛋白以包涵体的形式高效表达,在非变性条件下获得较高纯度的重组蛋白。生物信息学软件预测C7orf69蛋白(含信号肽)含有PKC、CK2磷酸化位点和N-十四烷基化位点。结论成功的对分泌蛋白C7orf69进行了基因克隆、表达与初步纯化,为其进一步的功能研究奠定了坚实的基础。

Objective To clone, express and purify recombinant protein C7orf69 in vitro, and to analyze its bioinformatics by prediction software. Methods Gene C7orf69 was amplified by polymerase chain reaction （PCR） and inserted into prokaryotic expression plasmid. Then the plasmid was transformed into E. coli BL21, induced by IPTG, identified by SDS-PAGE and Western blot, and purified under native or denaturing conditions by Ni-NTA resin. Results The right sequenced gene C7orf69 cotaining a natural variation was obtained, and the recombinant protein was highly expressed in the form of inclusion body. Purified recombinat protein was obtained under native conditions by Ni-NTA resin. The bioinformatics analysis by prediction software indicates that there are PKC and CK2 phosphorylation sites and a myristylation site in the protein C7orf69 including the signal peptide. Conclusions C7orf69 was successfully cloned, expressed and purified. These jobs lay solid foundation for the further study of its functions.