人E1A激活基因阻遏子的表达、抗体制备及生物学活性分析

目的:构建人E1A激活基因阻遏子(humancellularrepressorofE1A-stimulatedgene,hCREG)基因的原核表达载体,纯化重组的hCREG融合蛋白并制备兔抗hCREG抗体。探讨hCREG蛋白在人血管平滑肌细胞克隆株(HITASY)中的表达、定位。方法:用PCR技术,扩增hCREG并构建pGEX-4T-1/hCREG原核表达载体。诱导表达重组谷胱甘肽巯基转移酶(glutathioneS-transferase,GST)-hCREG融合蛋白。以GST-hCREG为抗原,制备兔抗人GST-hCREG的抗血清,并通过蛋白A和GST亲和层析纯化。用ELISA和Westernblot法检测抗体的效价和特异性;用免疫荧光染色法检查去血清培养前后HITASY细胞中hCREG蛋白的表达及定位;用BrdU染色观察分泌型hCREG蛋白对HITASY细胞增殖的影响。结果:经鉴定证实构建的重组质粒正确。诱导后pGEX-4T-1/hCREG菌株可表达大量的GST-hCREG融合蛋白,层析纯化后其纯度达90%。Westernblot检测表明,纯化的兔抗hCREG抗体可与hCREG蛋白特异性结合。ELISA测得该抗体的效价>1∶105。免疫荧光染色检测显示,去血清培养诱导的HITA-SY细胞中hCREG蛋白的表达增加且定位在核周围。BrdU染色表明,hCREG可明显抑制HITASY细胞增殖。结论:成功地获得兔抗hCREG抗体。证实去血清培养的血管平滑肌细胞中CREG蛋白的表达上调。表达的hCREG蛋白可抑制HI-TASY细胞增殖,提示hCREG可能参与调控血管平滑肌细胞的增殖与分化。

Expression,antibody production and bioactivity detection of cellular repressor of E1A-stimulated gene

AIM: To obtain the human cellular repressor of E1A-stimulated gene (hCREG) protein and the polyclonal antibody against the hCREG, and to further observe the expression and localization of hCREG protein in human internal thoracic artery cells (HITASY). METHODS: The hCREG cDNA was amplified by PCR and cloned into the pGEX-4T-1. Glutathione-S-transferase (GST)-hCREG fusion protein was expressed in E.coli BL21 and was used to immunize rabbits to obtain anti-hCREG serum, which was purified by protein A and GST immobilized on glutathione-Sepharose beads. The titer and specificity of polyclonal antibodies were determined by ELISA and Western blot analysis. The expression and localization of hCREG protein were detected with immunofluorescence staining in HITASY cells after serum removal. The proliferation of HITASY cells affected by hCREG protein was examined by means of BrdU stain. RESULTS: It was confirmed that hCREG cDNA was correctly inserted into the vector by endonuclease digesting and DNA sequencing. The expressed GST-hCREG protein was purified by the gel-filtration and its purity was up to about 90%. The anti-hCREG polyclonal antibody was specific with high titer (>1:10(5)) and the hCREG protein was expressed in a perinuclear pattern in HITASY cells after serum deprivation. It was also observed that the proliferation of HITASY cells was obviously inhibited by hCREG protein. CONCLUSION: The hCREG protein was highly expressed in HITASY cells and inhibited HITASY cell proliferation, suggesting that it may be involved in the process of proliferation and differentiation of vascular smooth muscle cells.