小亚璃眼蜱CPL截短基因的多克隆抗体制备及其应用研究

Preparation and application of polyclonal antibody of H.anatolicum CPL truncated gene

组织蛋白酶L(CPL)是半胱氨酸蛋白水解酶,属于木瓜蛋白酶家族,参与蜱对血红蛋白降解与消化,是一种潜在的抗蜱疫苗候选抗原。为了制备小亚璃眼蜱(H.anatolicum)CPL多克隆抗体,并对其初步应用,本研究利用PCR方法扩增CPL截短基因(HanCPL)后构建pET-28a-HanCPL和pEGFP-C3-HanCPL质粒,pET-28aHanCPL经IPTG诱导后通过SDS-PAGE检测重组HanCPL蛋白(rHanCPL)的表达,结果显示,正确构建了pET-28a-HanCPL和p EGFP-C3-HanCPL质粒;rHanCPL以包涵体形式表达,分子量大小约为27 ku,可以被兔抗His tag抗体特异性识别;利用亲和层析柱和尿素透析法纯化rHanCPL后经western blot鉴定纯化效果,并测浓度后免疫新西兰兔,制备rHanCPL多克隆抗体,经western blot检测rHanCPL的反应性,采用间接ELISA测定多克隆抗体的效价。结果显示,rHanCPL纯化效果较好,浓度为3 mg/mL;rHanCPL可以与其多克隆抗体发生特异反应,且制备的r HanCPL多克隆抗体效价高达1:204800,可用于后续试验。利用制备的rHanCPL多克隆抗体作为一抗,通过免疫组化试验检测天然CPL在H.anatolicum中的定位,结果显示,天然CPL蛋白可以被rHanCPL多克隆抗体特异性识别,且其定位于H.anatolicum中的肠上皮细胞;另外将pEGFP-C3-HanCPL质粒转染至HEK-293T中,以制备的rHanCPL多克隆抗体作为一抗,使用western blot和IFA方法检测重组蛋白(EGFP-HanCPL)的表达及定位情况,结果显示EGFP-HanCPL蛋白也可与rHanCPL多克隆抗体特异性结合,其分子量约为46 ku,其主要定位于细胞质。本研究利用表达的rHanCPL制备了r HanCPL多克隆抗体,并初步将该多克隆抗体用于CPL的相关研究,为后续抗蜱疫苗蛋白抗原的筛选提供参考依据。

Cathepsin L(CPL)is a cysteine proteinase and belongs to the papain family.It is involved in midgut digestion and oocyte synthesis in tick physiology,and is a potential candidate antigen for anti-tick vaccines.Here,we prepared polyclonal antibody from the expression of H.anatolicum prokaryotic recombinant protein(r Han CPL)and explored the preliminary application of r Han CPL polyclonal antibody.In this study,the CPL truncated gene was amplified by PCR method and p ET-28a-Han CPL and p EGFP-C3-Han CPL plasmids were constructed.After p ET-28a-Han CPL was induced by IPTG,the expression of r Han CPL was analyzed and identified by SDS-PAGE and western blot.Affinity chromatography column and urea dialysis method were employed to purify r Han CPL and experimental rabbits were immunized to prepare r Han CPL polyclonal antibody.r Han CPL polyclonal antibody was used as the primary antibody for immunohistochemistry to detect the localization of natural CPL in H.anatolicum.The p EGFP-C3-Han CPL plasmid was transfected into human embryonic kidney cells(HEK-293T).Western blot and IFA methods were used to detect the expression and localization of EGFP-Han CPL,using r Han CPL polyclonal antibody as the primary antibody.The results of western blot and IFA experiments showed that EGFP-Han CPL protein could specifically bind to r Han CPL polyclonal antibody,with a molecular weight of about 46ku,and was mainly located in the cytoplasm.In this study,the r Han CPL polyclonal antibody was prepared by using the expressed r Han CPL protein,and the polyclonal antibody was preliminarily used in CPL related research to provide a reference for the subsequent screening of the recombinant protein antigens of the anti-tick vaccines.