不同种属TRIM5α B30.2基因克隆与原核表达及纯化的研究

Cloning,expression and purification of TRIM5α B30.2 variant proteins

目的对人、恒河猴和非洲绿猴的TRIM5α B30.2基因进行克隆、原核表达及纯化。方法用PCR技术从人、恒河猴、非洲绿猴TRIM5α全基因序列中扩增B30.2片段,约660 bp,翻译成相对分子质量约为23×103的蛋白质。将测序鉴定过的不同种属TRIM5α B30.2基因克隆到原核表达载体A64上,以可溶的形式在大肠埃希菌BL21(DE3)中高效表达,经TEV酶切、纯化后,用SDS-PAGE方法鉴定表达蛋白。结果获得了高纯度可溶的TRIM5α B30.2蛋白,纯度可达90%以上。结论成功的获得不同种属TRIM5α B30.2蛋白,为蛋白结构的研究奠定基础。

Objective To achieve the high purity of recombinant TRIM5α B30.2 variant proteins.Methods The 660 bp fragments of TRIM5α B30.2 gene variants were amplified by PCR from the full length gene of human,rhesus and African green monkey TRIM5α variants.The recombinant vectors of A64-TRIM5α B30.2 were constructed with a prokaryotic expression plasmid（A64）and the TRIM5α B30.2 variant gene fragments.The variant proteins of TRIM5α B30.2 were expressed in Eserichia coli BL21（DE3） as soluble proteins.A six-histidine（His6） tag at the C-terminus of recombinant proteins was used to facilitate the protein purification.Results The recombinant constructs with TRIM5α B30.2 variant genes were identified by enzymes digestion.The size of 23 kD recombinant proteins was confirmed by SDS-PAGE.The purity of TRIM5α B30.2 variant proteins was above 90%.The assays of functional testing for TRIM5α B30.2 variant proteins were under process.Conclusion The high purity of TRIM5α B30.2 variant proteins was achieved with recombinant technology.