黄颡鱼Cu稳态相关基因ccs和cox17的原核表达及多克隆抗体制备

PROKARYOTIC EXPRESSION AND POLYCLONAL ANTIBODY PREPARATION OF CU HOMEOSTASIS-RELATED GENES CCS AND COX17 IN YELLOW CATFISH TACHYSURUS FULVIDRACO

为深入研究黄颡鱼Tachysurus fulvidraco Cu稳态调控机制,从黄颡鱼cDNA中克隆获得了810和192 bp的ccs (Pfccs)和cox17 (Pfcox17)基因编码序列,并成功构建了p ET32a(+)-Pfccs和p ET32a(+)-Pfcox17重组表达载体;转化重组质粒到E. coli Rosetta (DE3)感受态细胞中,以IPTG(异丙基-β-D-硫代半乳糖苷)作为诱导剂诱导表达,通过SDS-PAGE鉴定重组蛋白表达,发现PfCCS蛋白主要在沉淀中表达, PfCOX17蛋白主要在上清中表达;分别采用包涵体和亲和层析纯化的方法得到了纯度较高的重组PfCCS和PfCOX17蛋白;以纯化得到的PfCCS和PfCOX17蛋白免疫Balb/C小鼠3次,制备多克隆抗体。通过Western Blot鉴定了PfCCS和PfCOX17抗血清可以分别特异性识别重组PfCCS和PfCOX17蛋白,也可以分别特异性识别黄颡鱼内源性CCS和COX17蛋白。通过Western Blot对黄颡鱼CCS和COX17蛋白在鳃、心脏和肝脏中的分布情况进行检测,发现黄颡鱼CCS和COX17蛋白在肝脏中具有较高的表达水平, CCS蛋白在鳃中的表达水平最低,而COX17蛋白在鳃和心脏中的表达水平都很低。综上所述,研究成功制备了黄颡鱼CCS和COX17蛋白的多克隆抗体,为深入研究CCS和COX17蛋白在Cu转运中的作用及黄颡鱼Cu稳态调控机制奠定基础。

CCS(Copper chaperone for superoxide dismutase)and COX17(Cytochrome c oxidase copper chaperone 17)proteins are two common copper(Cu)chaperone proteins that play an important role in the regulation of Cu homeostasis.In order to further investigate the regulatory mechanism of Cu homeostasis in yellow catfish Tachysurus fulvidraco,810 and 192 bp coding sequences of T.fulvidraco ccs(Pfccs)and T.fulvidraco cox17(Pfcox17)genes were cloned from the cDNA of yellow catfish,and the recombinant expression vectors pET32a(+)-Pfccs and pET32a(+)-Pfcox17 were successfully constructed.Recombinant plasmids were transformed into E.coli Rosetta(DE3)competent cells,IPTG(Isopropyl-β-D-thiogalactopyranoside)was used as an inducer to induce expression,and the expression of recombinant proteins was identified by SDS-PAGE.We found that PfCCS protein was mainly expressed in the sediment,and PfCOX17 protein was mainly expressed in the supernatant.The purified recombinant PfCCS and PfCOX17 proteins were obtained by inclusion and affinity chromatography,respectively.Polyclonal antibodies were prepared by immunizing Balb/C mice three times with purified PfCCS and PfCOX17 proteins.Western Blot analysis showed that PfCCS and PfCOX17 antisera could specifically recognize recombinant PfCCS and PfCOX17 proteins,and could also specifically recognize endogenous CCS and COX17 proteins of yellow catfish,respectively.The distribution of CCS and COX17 proteins in gill,heart and liver was detected by Western Blot,and the expression levels of CCS and COX17 proteins were higher in liver,the expression levels of CCS protein in gill were the lowest,and the expression levels of COX17 protein in gill and heart were very low.In conclusion,this study successfully prepared polyclonal antibodies that can specifically recognize recombinant PfCCS and PfCOX17 proteins,and murine PfCCS and PfCOX17 antibodies can also be well used in the Western Blot detection of CCS and COX17 protein of yellow catfish,which laid a foundation for further study on the roles of CCS and COX17 protein in Cu transport and regulatory mechanism of Cu homeostasis in yellow catfish.