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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 clone, prokaryotic and purify TRIM5α B30.2 gene of human, rhesus macaque and African green monkey. Methods The B30.2 fragment of about 660 bp was amplified from the full length TRIM5α gene of human, rhesus monkey and African green monkey by PCR and translated into a protein with a relative molecular mass of about 23 × 103. The TRIM5α B30.2 gene of different species identified by sequencing was cloned into the prokaryotic expression vector A64 and expressed in soluble form in Escherichia coli BL21 (DE3). After digested with TEV and purified by SDS- PAGE method to identify the expressed protein. The results obtained high purity soluble TRIM5α B30.2 protein, purity of up to 90%. Conclusion The TRIM5α B30.2 protein from different species was successfully obtained, which laid the foundation for the study of protein structure.