草鱼IFNa和IFNd重组蛋白表达、纯化及单克隆抗体制备

为系统研究草鱼I型干扰素的合成、分泌和免疫功能，本研究在大肠杆菌中表达并提纯了草鱼IFNa（CiIFNa）和IFNd（CiIFNd）重组蛋白。将CiIFNa和CiIFNd成熟肽分别克隆到pET-21d或pEHISTEVb表达载体上，并转化到大肠杆菌中；IPTG诱导表达得到CiIFNa和CiIFNd成熟肽的包涵体，经过盐酸胍变性、蛋白复性和浓缩后，利用AKTA分子筛层析获得了纯度较高的重组蛋白。用重组蛋白免疫小鼠，通过PEG法诱导得到杂交瘤细胞；将稳定分泌抗体的阳性细胞株的细胞悬液注射入小鼠腹腔，制备腹水抗体并进行纯化。本研究纯化了草鱼CiIFNa和CiIFNd各2株抗体，并采用SDS-PAGE、ELISA、Western blot和免疫荧光法对其进行了较全面的鉴定。研究结果表明CiIFNa和CiIFNd单克隆抗体特异性好、效价高，能够特异识别在大肠杆菌和真核细胞中表达的重组蛋白，且不存在CiIFNa和CiIFNd分子间的交叉识别。本研究制备的单克隆抗体为深入研究草鱼干扰素的细胞来源和蛋白表达规律奠定基础。

Expression of recombinant proteins and preparation of monoclonal antibodies for IFNa and IFNd in grass carp (Ctenopharyngodon idella)

Abstract: In order to understand the synthesis, secretion and immune functions of type I interferons in Ctenopharyngodon idella, CiIFNa and CiIFNd were expressed in the Escherichia coli (E. coli) cells and purified. The mature peptides of CiIFNa and CiIFNd were cloned into pET-21d and pEHISTEVb expression vectors, respectively, and transformed in E. coli Rosetta cells. The recombinant proteins were expressed as inclusion bodies after IPTG induction. Following denaturation with guanidine hydrochloride, renaturation and concentration, the recombinant proteins were purified by size exclusion chromatography and used for immunization of mice. Hybridoma cells were obtained using the PEG method and injected into the abdominal cavity of mice to generate ascites. Two monoclonal antibodies of CiIFNa and CiIFNd were purified, respectively, and characterized by SDS-PAGE, ELISA, Western blot and immunofluorescence assay. It has been shown that the monoclonal antibodies produced had good specificity and high titers against the antigens and could specifically recognize recombinant proteins expressed in E. coli and eukaryotic cells. The CiIFNa antibodies did not react with CiIFNd and vice versa. Taken together, the CiIFNa and CiIFNd monoclonal antibodies prepared in this study laid the basis for further in-depth study of the cellular sources and protein expression profiles of IFNs in grass carp.