中国水产科学2017，Vol.24Issue(5)：970-976,7.DOI:10.3724/SP.J.1118.2017.16343
淇河鲫IL-8原核表达系统的构建及多克隆抗体的制备与鉴定
Construction of prokaryotic expression system for IL-8 of Carassius auratus var.Qihe and preparation of polyclonal antibody
摘要
Abstract
Interleukin-8 (IL-8),a chemokine that participates in the early inflammatory process,plays a key role in mediating resistance to infection in animals.However,there has been no information about IL-8 at the protein level for characterizing the regulatory role of this molecule during the immune response in fish.In this work,we constructed a prokaryotic expression system and prepared a polyclonal antibody against IL-8 of Qihe crucian carp (Carassius auratus var.Qihe).The coding region without a signal peptide sequence was first amplified and cloned into pET-32a(+),a prokaryotic expression plasmid.The recombinant plasmid was then transformed into Escherichia coli Rosetta.A soluble fusion protein was expressed after induction by isopropyl β-D-1-thiogalactopyranoside.The purified protein was detected as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis,with a predicted molecular mass of 27.8 kD,suggesting that the prokaryotic expression system for IL-8 of Qihe crucian carp was successfully constructed.The purified recombinant protein was used as an immunogen to raise polyclonal antibodies in New Zealand rabbits.The serum antibody titer reached 1:105,as detected by indirect enzyme-linked immunosorbent assay.The antibody was purified by affinity chromatography and had a high binding activity and specificity for the recombinant protein antigen,as measured by Western Blot.Immunohistochemistry results showed that the polyclonal antibody can also specifically bind to endogenous IL-8 in crucian carp tissues.Comparing the results of immunohistochemistry with that of fluorogenic quantitative polymerase chain reaction,the expression of IL-8 was consistent between the protein and mRNA levels,and its expression ranked among tissues as follows:muscle > spleen > head kidney > intestine.This study provides a material basis for further research on the role of IL-8 in the immune response of Carassius auratus var.Qihe.关键词
淇河鲫/白细胞介素-8/原核表达系统/多克隆抗体/组织表达/免疫组化Key words
Carassius auratus var.Qihe/interleukin-8/prokaryotic expression system/polyclonal antibody/tissue expression/immunohistochemistry分类
农业科技引用本文复制引用
王俊丽,陈金顶,卢荣华,雒燕婷,聂国兴..淇河鲫IL-8原核表达系统的构建及多克隆抗体的制备与鉴定[J].中国水产科学,2017,24(5):970-976,7.基金项目
国家自然科学基金项目(31372545) （31372545）
河南省高校科技创新团队支持计划项目(14IRTSTHN013) （14IRTSTHN013）
河南省科技计划基金项目(142300410159) （142300410159）
河南省教育厅科学技术重点研究项目(14A180007) （14A180007）
河南省自然科学基金项目(162300410165). （162300410165）
