【摘要】 目的:构建新基因CTRP4的原核表达载体,在大肠杆菌中表达并纯化rhCTRP4-his蛋白,制备和鉴定小鼠抗人CTRP4多克隆抗体,为进一步研究人类新基因CTRP4的生物学功能奠定基础。方法:从pcDNA3.1-myc/his(-)B-hCTRP4重组载体中通过PCR的方法扩增CTRP4基因ORF中不含信号肽的目的基因片段,将得到的片段双酶切定向插入pET-32a中,构建原核表达载体pET-32a-hCTRP4。构建好的质粒在E.coli BL21(DE3)中进行诱导、表达,通过镍亲和层析柱纯化融合蛋白,SDS-PAGE电泳分析蛋白纯度。将pcDNA3.1-myc/his(-)B-hCTRP4重组质粒和纯化好的融合蛋白依次免疫BALB/c小鼠,制备多克隆抗体。并对抗体进行纯化、效价测定以及特异性鉴定。结果:DNA测序证实构建的pET-32a-hCTRP4重组表达载体含有hCTRP4编码序列,其序列比对分析与GenBank中公布序列一致,质粒在E.coli中表达相对分子质量(Mr)为35 000的目的蛋白,SDS-PAGE电泳分析纯度为95%以上。ELISA法检测抗体效价为1∶20 000,Westernblot、免疫荧光细胞化学和免疫组化分析显示抗体能特异性识别重组hCTRP4以及天然状态hCTRP4蛋白。结论:成功构建了hCTRP4蛋白的原核表达载体,并获得较高纯度的融合蛋白,制备了高效价、高特异性的多克隆抗体。更多还原

【Abstract】 AIM: To further investigate the biological function of human novel gene CTRP4 by constructing the prokaryotic expression vector of human CTRP4,inducing the expression of and purifying hCTRP4-his protein in E.coli,and preparing polyclonal antibody against human CTRP4.METHODS: Human CTRP4 gene was amplified by PCR,digested with enzymes,and subcloned into a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-hCTRP4.The pET-32a-hCTRP4 was transformed into E.coli BL21(DE3).The hCTRP4-his fusion protein was induced by IPTG,purified by Ni-NTA purification system,and analyzed by SDS-PAGE.The recombinant vector pcDNA3.1-myc/his(-)B-hCTRP4 expressing full-length human CTRP4 and purified prokaryotic protein hCTRP4 were used to immunize BALB/c mice to produce polyclonal antibody.The anti-serum was purified and the characteristics of the antibody were identified by ELISA,Western blotting,immunofluorescence cytochemistry and immunohistochemistry.RESULTS: The prokaryotic expression vector of pET-32a-hCTRP4 was constructed successfully.hCTRP4-his fusion protein was expressed in E.coli BL21(DE3) after IPTG induction.The titer of the anti-serum reached 1∶ 20 000,and its specificity was proved by Western blotting.The results of immunofluorescence cytochemistry and immunohistochemistry indicated that CTRP4 was mainly localized in the cytoplasm of hepatic cells.CONCLUSION: hCTRP4-his fusion protein can be successfully expressed in E.coli.A specific polyclonal antibody against human CTRP4 has been successfully prepared.更多还原