Tan Wei-feng, Wang Lan-lan, Li Qi, Luo Yang, Na Da-xiang, Ma Zhuang, Wang Lu
Center for Human Disease Genomics, Peking University, Beijing, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Jun;28(6):614-7.
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.
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.
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.
hCTRP4-his fusion protein can be successfully expressed in E.coli. A specific polyclonal antibody against human CTRP4 has been successfully prepared.
通过构建人CTRP4的原核表达载体，在大肠杆菌中诱导表达并纯化hCTRP4-his蛋白，制备抗人CTRP4的多克隆抗体，进一步研究人新基因CTRP4的生物学功能。
采用PCR扩增人CTRP4基因，酶切后亚克隆至带his标签的原核表达载体，构建重组质粒pET-32a-hCTRP4。将pET-32a-hCTRP4转化至大肠杆菌BL21(DE3)。用IPTG诱导表达hCTRP4-his融合蛋白，经Ni-NTA纯化系统纯化，SDS-PAGE分析。用表达全长人CTRP4的重组载体pcDNA3.1-myc/his(-)B-hCTRP4和纯化的原核蛋白hCTRP4免疫BALB/c小鼠制备多克隆抗体。纯化抗血清，通过ELISA、Western印迹、免疫荧光细胞化学和免疫组织化学鉴定抗体特性。
成功构建pET-32a-hCTRP4原核表达载体。IPTG诱导后，hCTRP4-his融合蛋白在大肠杆菌BL21(DE3)中表达。抗血清效价达1:20 000，Western印迹证明其特异性。免疫荧光细胞化学和免疫组织化学结果表明CTRP4主要定位于肝细胞胞质。
hCTRP4-his融合蛋白可在大肠杆菌中成功表达。已成功制备抗人CTRP4的特异性多克隆抗体。
