摘要
干扰素基因刺激因子(STING)是天然免疫信号通路环鸟苷酸-腺苷酸合成酶(cGAS)-STING中重要的衔接蛋白,它能够识别细胞内第二信使环磷酸鸟苷-腺苷(c GAMP),激活I型干扰素(IFN-I)的表达。为了制备猪STING(pSTING)多克隆抗体(ppSTING)并对其初步应用,本研究利用PCR方法扩增pSTING基因构建重组表达质粒pGEX-6p1-pSTING,转化大肠杆菌BL21(DE3)并经IPTG诱导后经SDS-PAGE和western blot方法鉴定GSTpSTING蛋白(rpSTING)的表达;利用谷胱甘肽琼脂糖亲和层析方法纯化rpSTING并免疫新西兰大白兔制备ppSTING,经Protein G亲和层析介质纯化后分别采用SDS-PAGE鉴定纯化的ppSTING。结果显示rpSTING以可溶形式表达,分子质量约为52.5 ku。SDS-PAGE结果显示,制备的ppSTING包含重、轻链且纯度较好。将ppSTING分别应用于western blot、间接免疫荧光试验(IFA)检测外源表达及CRL-2843细胞中内源表达的pSTING;将其应用于Co-IP试验检测ppSTING与外源表达pSTING的相互作用;将其应用于激光共聚焦试验检测cGAMP刺激后的PK-15细胞中内源表达的pSTING与AP-1在该细胞中的共定位;将其应用于western blot检测ASFV感染后PAMs中的pSTING蛋白。Western blot和IFA结果显示,制备的ppSTING能特异性地识别HEK293T细胞中过表达及CRL-2843细胞中内源表达的pSTING蛋白;Co-IP结果显示,ppSTING与pSTING蛋白存在相互作用;激光共聚焦试验结果显示,ppSTING可用于检测PK-15中内源表达的pSTING蛋白且在cGAMP刺激后的PK-15细胞中的pSTING能够与AP-1定位于高尔基体。Western blot结果显示,ppSTING能够与未感染ASFV的PAMs中内源表达的pSTING蛋白反应,且在ASFV感染PAMs 24 h后pSTING蛋白的表达量增加。上述结果表明,本研究制备了ppSTING并且证实其可以用于宿主天然免疫方面的应用研究。本研究为探究pSTING蛋白的生物学功能以及cGAS-STING信号通路奠定了基础。
Interferon gene stimulating factor(STING) is an important adaptor protein in the innate immune signal pathway of cyclic guanylate-adenylate synthase(c GAS)-STING, which can recognize the second messenger cyclic guanosine monophosphateadenosine(c GAMP) to activates the expression of type I interferon(IFN-I). In order to prepare polyclonal antibody(pp STING)against porcine STING(p STING) and its preliminary application, in this study, p STING gene was amplified using polymerase chain reaction(PCR) method, and recombinant expression plasmid p GEX-6 p1-p STING was constructed, then transformed into E. coli BL21(DE3) and induced by IPTG. The expression of GST-p STING protein(rp STING) was identified by SDS-PAGE and western blot. After purified by glutathione agarose affinity chromatography, Rp STING was used to immunize New Zealand white rabbits to prepare pp STING. The pp STING was purified by protein G affinity chromatography media and identified by SDS-PAGE.The results showed that rp STING was expressed in a soluble form with a molecular mass of about 52.5 ku. SDS-PAGE analysis showed that the prepared pp STING contained heavy and light chains with good purity. Pp STING was used to detect the exogenously expressed p STING and the endogenous expression of p STING in CRL-2843 cells by western blot and indirect immunofluorescence assay(IFA), respectively;the interaction between pp STING and exogenously expressed p STING was evaluated by Co-IP test;the co-localization of endogenously expressed p STING and AP-1 in PK-15 cells stimulated by c GAMP was detected by laser confocal test;and the p STING protein in PAMs after ASFV infection was analyzed by western blot. Western blot and IFA results showed that the prepared pp STING can specifically recognize the over-expressed p STING protein in HEK293 T cells and the endogenously expressed p STING protein in CRL-2843 cells;Co-IP analysis showed that pp STING could interact with p STING protein;pp STING can be used to detect the endogenously expressed p STING protein in PK-15 and that p STING in PK-15 cells stimulated by c GAMP can be localized to the Golgi apparatus with AP-1 based on the laser confocal test;as well as pp STING could react with the p STING protein endogenously expressed in PAMs not infected with ASFV, and the expression of p STING protein increased 24 hours after ASFV infection of PAMs based on western blot analysis. These results demonstrated that the prepared pp STING in this study can be used in the application research of host innate immunity. This study laid the foundation for exploring the biological function of p STING protein and the c GAS-STING signaling pathway.
作者
薛梦迪
许文杰
向志达
黄丽
翁长江
XUE Meng-di;XU Wen-jie;XIANG Zhi-da;HUANG Li;WENG Chang-jiang(Division of Fundamental Immunology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,State Key Laboratory of Veterinary Biotechnology,Harbin 150069,China;College of Life Sciences,Yangtze University,Jingzhou 434025,China;College of Animal Science and Technology,Yangtze University,Jingzhou 434025,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第8期860-866,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31941002)。
关键词
pSTING蛋白
原核表达
多克隆抗体
porcine STING protein
prokaryotic expression
polyclonal antibody
