鳜AKT2在传染性脾肾坏死病毒增殖中的作用

Role of Siniperca chuatsi AKT2 in proliferation of infectious spleen and kidney necrosis virus

【目的】研究鳜AKT2(ScAKT2)在传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)增殖中的作用,为鳜ISKNV防控提供参考。【方法】克隆ScAKT2基因开放阅读框,构建原核表达载体,进行原核表达及蛋白纯化。用纯化后的ScAKT2重组蛋白免疫日本大耳兔后制备多克隆抗体,用间接ELISA法检测抗体的效价,用间接免疫荧光和Western blot法测定抗体的特异性。克隆鳜AKT2基因,构建过表达载体pCMV-EGFP-ScAKT2,将其转染至CPB细胞系中构建ScAKT2过表达细胞系,用荧光显微镜检测转染效率,用qRT-PCR法及Western blot法分别测定ScAKT2 mRNA水平和蛋白水平的表达,以鉴定ScAKT2过表达细胞系是否构建成功。将ISKNV接种至ScAKT2过表达细胞系中,用qPCR法及Western blot法分别测定ISKNV病毒拷贝数及ISKNV-MCP蛋白的表达,用qRT-PCR法测定ScIRF3、ScIRF7、ScMx、ScIL8、ScTRAF2和ScTRAF3等先天免疫因子mRNA水平的表达情况。【结果】PCR扩增获得1400 bp的AKT2 ORF片段,成功构建了原核表达质粒,诱导表达出72 ku的重组蛋白。成功制备了ScAKT2多克隆抗体,抗体效价达1∶256000,纯化后质量浓度约为10 mg/mL。成功构建了pCMV-EGFP-ScAKT2过表达载体和过表达ScAKT2的CPB细胞系,过表达ScAKT2 CPB细胞可极显著抑制ISKNV的增殖,能明显上调ScIRF3、ScIRF7、ScMx、ScIL8、ScTRAF2和ScTRAF3等先天免疫因子mRNA的表达水平。【结论】ScAKT2通过上调先天免疫因子的表达抑制ISKNV的增殖,为鳜ISKNV防控提供了新的潜在靶点。

【Objective】This study investigated the role of Siniperca chuatsi AKT2(ScAKT2)in the proliferation of infectious spleen and kidney necrosis virus(ISKNV).【Method】The ScAKT2 gene open reading frame was cloned,and the prokaryotic expression vector was constructed for prokaryotic expression and protein purification.After purification,polyclonal antibodies were prepared after immunizing Japanese big ear rabbits with ScAKT2 recombinant protein,ELISA method was used to determine antibody titer,and IFA method and Western blot method were used to detect antibody specificity.The AKT2 gene was also cloned to construct overexpression vector pCMV-EGFP-ScAKT2 transfected into CPB cell lines to construct overexpression cell lines.Fluorescence microscope was used to examine the transfection efficiency of the eukaryotic expression vector,and the qRT-PCR method and Western blot method were used to determine the expression of ScAKT2 mRNA and protein levels to identify whether the ScAKT2 overexpressing cell line was successfully constructed.Inoculating ISKNV into the ScAKT2 overexpressing cell line,qPCR method and Western blot method were used to determine ISKNV virus copy number and ISKNV-MCP protein expression,respectively.The qRT-PCR method was used to examine the mRNA expression of innate immune factors including ScIRF3,ScIRF7,ScMx,ScIL8,ScTRAF2 and ScTRAF3.【Result】PCR amplification obtained 1400 bp of AKT2 ORF fragments and successfully constructed prokaryotic expression plasmids to induce the expression of 72 ku of recombinant proteins.ScAKT2 polyclonal antibody was successfully prepared with a titer of 1∶256000 and a concentration of about 10 mg/mL after purification.CPB cell lines pCMV-EGFP-ScAKT2 overexpression vectors and overexpression ScAKT2 were successfully constructed.Overexpression of ScAKT2 CPB cells extremely significantly inhibited ISKNV proliferation and significantly upregulated the mRNA expression levels of innate immune factors including ScIRF3,ScIRF7,ScMx,ScIL8,ScTRAF2 and ScTRAF3.【Conclusion】ScAKT2 inhibited the proliferation of ISKNV by up-regulating the expression of innate immune factors.This study provides a new potential target for the prevention and control of ISKNV.