抗小鼠白细胞介素-1α多克隆抗体的制备与应用

[Preparation and application of polyclonal antibody against mouse IL-1α].

作者信息

Lin Dan-Dan, Liu Chun-Liang, Bao Guang-Ming, Wu Kang, Shan Lin, Liu Hai-Yan

机构信息

Laboratory of Cellular and Molecular Tumor Immunology, Medical College of Soochow University, Suzhou 215123, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2011 Sep;27(9):989-92.

PMID:21906474

Abstract

AIM

To construct a recombinant plasmid encoding mouse IL-1α (mIL-1α), express and purify mIL-1α protein, and prepare its polyclonal antibody.

METHODS

The cDNAs were obtained from the spleen cells of BALB/c mice and the full length of mIL-1α gene was amplified by RT-PCR. Then the mIL-1α gene was inserted into a prokaryotic expression vector pET32a(+) and the resulting recombinant plasmid was transformed into E.coli BL21(DE3). After auto-induction, the mIL-1α protein was expressed and purified by electro-elution. An anti-mIL-1α polyclonal antibody was raised in New Zealand rabbits after immunization with the purified mIL-1α and the titer was determined by ELISA. The specificity of the polyclonal antibody was identified by Western blot and flow cytometry.

RESULTS

The recombinant prokaryotic expression vector pET32a(+)-IL-1α was successfully constructed, and the mIL-1α protein was expressed and purified. ELISA showed the titer of the anti-mIL-1α serum was 1:25 600. Western blot and flow cytometry demonstrated the high specificity of the polyclonal antibody to IL-1α.

CONCLUSION

The rabbit anti-mIL-1α polyclonal antibody with high titer and specificity has been prepared after immunization with the purified mIL-1α protein, facilitating further functional studies of IL-1α.

摘要

目的

构建编码小鼠白细胞介素-1α（mIL-1α）的重组质粒，表达并纯化mIL-1α蛋白，制备其多克隆抗体。

方法

从BALB/c小鼠脾脏细胞中获取cDNA，通过逆转录聚合酶链反应（RT-PCR）扩增mIL-1α基因全长。然后将mIL-1α基因插入原核表达载体pET32a(+)，所得重组质粒转化至大肠杆菌BL21(DE3)。经自动诱导后，表达mIL-1α蛋白并通过电洗脱法进行纯化。用纯化的mIL-1α免疫新西兰兔制备抗mIL-1α多克隆抗体，采用酶联免疫吸附测定（ELISA）法测定抗体效价。通过蛋白质免疫印迹法（Western blot）和流式细胞术鉴定多克隆抗体的特异性。