中国旱獭Ⅰ型干扰素受体β亚基克隆、表达及功能初步鉴定  

Preliminary identification of the cloning,expression,and function of Marmota himalayana typeⅠinterferon receptorβsubunit

作　　者：陶颖 杨东亮[2] 王宝菊[2] 刘艺 桂文甲 李智 樊和斌 TAO Ying;YANG Dongliang;WANG Baoju;LIU Yi;GUI Wenjia;LI Zhi;FAN Hebin(Department of Infectious Diseases,General Hospital of The Central Theater Command,Wuhan 430030,China;Department of Infectious Diseases,Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]中国人民解放军中部战区总医院感染科,武汉430030 [2]华中科技大学同济医学院附属协和医院感染科,武汉430030

出　　处：《临床肝胆病杂志》2024年第2期278-283,共6页Journal of Clinical Hepatology

基　　金：国家自然科学基金(3021170);国家重大基础研究项目(973)(2005CB522900)。

摘　　要：目的克隆中国旱獭Ⅰ型干扰素受体β亚基(mhIFNAR2)的基因,并进行抗体制备及功能鉴定。方法应用RT-PCR技术,从中国旱獭脾组织中扩增得到序列,克隆至原核表达载体p RSET-B,表达重组蛋白,电泳和Western Blot法鉴定;重组蛋白常规免疫BALB/c小鼠制备其胞外段多克隆抗体,免疫组化、免疫荧光和Western Blot法鉴定;再通过si RNA阻断的方法检测其功能。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果从mhIFNAR2扩增出149~1300 bp片段,其同源性在分析的种属中以土拨鼠最高,可达98.05%。成功地构建了表达胞外段mhIFNAR2_((50-181aa))蛋白的原核表达质粒,命名为pRSET-B.mhIFNAR2;其表达重组蛋白分子量27 kD,纯化后纯度约为95%,浓度约为160μg/mL。用纯化的重组蛋白常规免疫BALB/c小鼠后,获得1∶1000的特异性多克隆抗体,用免疫组化及免疫荧光可见细胞膜、细胞质有表达。合成的三条siRNA,其中有一条起始于277位点的siRNA(siRNA277)与空白对照及阴性对照相比,可以沉默目的基因的表达,并能减弱干扰素的信号通路(P值均<0.05)。结论获得mhIFNAR2的部分序列,成功地制备出抗mhIFNAR2胞外段多克隆抗体,该抗体有较高的效价和特异性,并能用于免疫组化、免疫荧光及Western Blot的检测。用siRNA277可以抑制目的基因的表达,并能阻断干扰素的信号通路。Objective To clone the gene of Marmota himalayana typeⅠinterferon receptorβsubunit(mhIFNAR2),and to perform antibody preparation and functional identification.Methods RT-PCR was used for amplification in the spleen tissue of Marmota himalayana to obtain the sequence,which was cloned to the prokaryotic expression vector pRSET-B to express the recombinant protein.Electrophoresis and Western blot were used for identification.BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody of its extracellular domain;immunohistochemistry,immunofluorescence assay,and Western Blot were used for identification,and the method of siRNA blockade was used to investigate its function.An analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for comparison between two groups.Results A fragment of mhIFNAR2(149—1300 bp)was obtained from spleen tissue,which showed the highest homology of 98.05%in marmot.A prokaryotic expression plasmid was successfully constructed for expression of the extracellular domain of the mhIFNAR2_((50-181aa))and was named pRSET-B.mhIFNAR2,and the recombinant protein expressed by this plasmid had a molecular weight of 27 kD,a purity of about 95%after purification,and a concentration of 160μg/mL.After BALB/c mice were immunized with the purified recombinant protein,1∶1000 specific polyclonal antibodies were obtained,and immunohistochemistry and immunofluorescence assay showed the expression in cell membrane and cytoplasm.Among the three siRNAs synthesized,the siRNA starting from the 277 locus(siRNA277)could silence the expression of target genes and weaken the interferon signaling pathway compared with the blank control group and the negative control group(both P<0.05).Conclusion The fragment of mhIFNAR2 is obtained,and the polyclonal antibody for the extracellular domain of mhIFNAR2 is successfully prepared,with relatively high titer and specificity,and can be used for immunohistochemis

关 键 词：受体 干扰素αβ 克隆 乙型肝炎 

分 类 号：R392[医药卫生—免疫学]