Su Wen-hui, Zhang Jie, Zhang Li-yan, Zong Zhi-hong, Zhang Zhe, Yu Bing-zhi
Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Sep;22(5):553-6.
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.
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.
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 plasmid 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.
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.
构建人新基因睾丸精子结合蛋白（tsbp）的真核表达载体，表达融合蛋白并通过亲和层析纯化重组蛋白。
从原核表达质粒pGEX - 5X - 1/tsbp中通过PCR扩增新基因tsbp，经DNA重组后构建真核表达载体pcDNA3.1/myc - His(-)B/tsbp。通过脂质体介导将该重组载体转染HEK293细胞后，采用RT - PCR、免疫荧光和Western印迹法检测融合蛋白的表达。利用固定化金属亲和层析（IMAC）从细胞裂解物中纯化融合蛋白His6 - tsbp，并通过SDS - PAGE和Western印迹法检测纯化效率。
DNA测序和限制性内切酶消化分析表明真核表达载体pcDNA3.1/myc - His(-)B/tsbp构建成功。将重组质粒转染HEK293细胞后，RT - PCR验证了tsbp mRNA的表达。免疫荧光检测结果为阳性，转染的HEK293细胞的Western印迹法可检测到融合蛋白。纯化的融合蛋白也可通过SDS - PAGE和Western印迹法检测到。
新基因tsbp成功克隆，并以His6融合蛋白的形式表达和纯化，这有助于进一步研究该睾丸精子结合蛋白的功能。
