人类新基因CTRP4的原核表达及多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of human novel gene CTRP4

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。

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 expres- sion vector of pET-32a-hCTRP4 was constructed successful- ly. 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. coil A specific polyclonal antibody against human CTRP4 has been successfully prepared.