兔白细胞介素-10基因的克隆、原核表达及其多克隆抗体的制备

Cloning,Prokaryotic Expression and Polyclonal Antibody Preparation of Rabbit IL-10 Gene

白细胞介素-10(IL-10)是一种多功能的细胞因子,参与免疫调节和抗炎反应。本研究旨在克隆并表达IL-10基因,以制备兔IL-10多克隆抗体。运用RT-PCR技术从新西兰白兔脾脏总RNA中扩增IL-10基因,将该基因亚克隆入原核表达载体pET-32a并转入大肠杆菌BL21(DE3)中诱导表达获得融合蛋白,融合蛋白经纯化后免疫豚鼠制备多克隆抗体,最后分别用间接ELISA和Western blot检测抗体的效价和特异性。结果表明:克隆得到了兔IL-10基因,大小为537bp,经核苷酸测序与GenBank已登录的基因序列(NM_001082045)相似性为99%;重组载体pET-IL-10构建成功;SDS-PAGE电泳结果显示融合蛋白成功表达;制备的多克隆抗体效价高达1∶56 000,且具有很高的特异性。成功实现了兔IL-10基因的原核表达,并制备了豚鼠抗兔IL-10多克隆抗体,为深入研究兔IL-10基因的功能奠定了基础。

Interleukin-10 （IL-10） has been recently identified as a multifunctional cytokine because of its close link with immune regulation and anti-inflammatory responses. The aim of this study was to clone and express rabbit IL-10 gene in order to prepare the polyclonal antibody against recombinant IL-10. The IL-10 gene of New Zealand white rabbit was amplified by RT-PCR from spleen total RNA, then was sub-cloned into prokaryotic expression vector pET-32a and expressed in E. coli BL21 （DE3） host cells. Fusion protein was purified and immunized into guinea pigs so as to prepare polyclonal antibody. Finally, the sensitivity and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot, respectively. As a result, rabbit IL-10 gene was cloned, and the obtained 537 bp fragment had 99% identities to the previously identified rabbit IL-10 gene （NM_001082045） at nucleotide levels; recombinant vector pET-IL-10 was successfully constructed; SDS-PAGE electrophoresis showed that the fusion protein was well expressed; the anti-IL-10 polyclonal antibody showed high sensitivity （1：56 000） and specificity. The above results indicate that IL-10 gene has been successfully cloned and expressed, and the guinea pig anti-rabbit IL-10 polyclonal antibody has been prepared by immunization with the purified recombinant IL-10 protein, which lays the foundation for further studies on rabbit IL-10 gene functions.