人睾丸精子结合蛋白-His6融合蛋白在真核细胞中的表达及亲和纯化

目的:构建含有人睾丸精子结合蛋白基因(testisspermbindingprotein,tsbp)的真核表达载体pcDNA3.1/mycHis(-)B/tsbp,并进行表达和纯化。方法:以克隆有tsbp全长cDNA序列的原核表达载体pGEX5X1/tsbp为模板,用PCR扩增tsbp,并通过DNA重组构建真核表达载体pcDNA3.1/mycHis(-)B/tsbp。以重组体稳定转染HEK293细胞后,应用RTPCR、Westernblot和细胞免疫荧光技术检测His6tsbp融合蛋白的表达。选择高表达量的细胞克隆,扩增并用Ni2 NTA金属亲和层析柱(IMAC)从细胞裂解液中纯化融合蛋白His6tsbp,纯化产物的纯度用SDSPAGE和Westernblot进行鉴定。结果:成功地构建了真核表达载体pcDNA3.1/mycHis(-)B/tsbp。以重组体转染HEK293细胞后,用RTPCR检测到tsbpmRNA的表达。细胞免疫荧光染色呈阳性反应,Westernblot检测到培养细胞中有融合蛋白His6tsbp的表达。经Ni2 NTA金属亲和层析柱分离纯化,得到纯化的重组蛋白His6tsbp。结论:构建了pcDNA3.1/mycHis(-)B/tsbp真核表达载体,并在HEK293细胞中表达和亲和层析纯化,为下一步的研究奠定了基础。

Fusion expression and affinity purification of a human novel gene tsbp in eukaryotic cells

AIM: To construct an eukaryotic expressing vector of the human novel gene testis sperm binding protein (tsbp), and to express fusion protein and purify the recombinant protein by affinity chromatography. METHODS: The novel gene tsbp was amplified by PCR from the prokaryotic expressing plasmid pGEX-5X-1/tsbp and an eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed after DNA recombination. After transfecting HEK293 cells with this recombinant vector via liposome mediation, the expression of the fusion protein was detected by RT-PCR, immunofluorescence and Western blot. Fusion protein His6-tsbp was purified from the cell lysis by immobilized metal affinity chromatography (IMAC) and the efficiency of purification was detected by SDS-PAGE and Western blot. RESULTS: DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp had been constructed successfully. After the recombinant plamid being transfected into HEK293 cells, RT-PCR verified the expression of tsbp mRNA. The result of immunofluorescence assay was positive and the fusion protein could be detected by Western blot of transfected HEK293 cells. The purified fusion protein could also be detected by SDS-PAGE and Western blot. CONCLUSION: The novel gene tsbp was successfully cloned, expressed and purified in the form of His6 fusion protein, which is helpful for further study of the function of this testis sperm binding protein.