大眼鰤鲈鱼真皮肿瘤病毒（WDSV）辅助基因orfA和orfC多抗的制备及初步应用

摘要

【Objective】The study was done to prepare polyclonal antibodies of walleye dermal sarcoma virus(WDSV)accessory genes orfA and orfC and to analyze the orfA and orfC proteins expressed in yeast and mammalian cells.【Method】In this research,the WDSV genes orfA and orfC were amplified from walleye dermal sarcoma virus complete genome and cloned into pET32a(+)to construct the prokaryotic expression vectors.Subsequently,Rosseta(DE3)competent cells were transformed with the constructed expression vectors,and the orfA and orfC recombinant proteins were prepared by induction with IPTG and purified by washing with 2 mol/L and 4 mol/L urea solution,successively.The purified proteins were used to immunize New Zealand rabbits using the method of hypodermic injection coupled with era vein booster immunization.The specificities of the polyclonal antibodies prepared were checked by Western blot analysis.Subsequently,the anti-orfA and anti-orfC rabbit serums were preliminarily applied to confirm the expression of orfA and orfC proteins in yeast and tumor cells.【Result】The prokaryotic expression vectors of orfA and orfC were constructed and the expression was inducted successfully.The orfA and orfC recombinant fusion proteins were in the form of inclusion bodies.The method of hypodermic injection coupled with era vein booster immunization improved the antibody titers effectively.The expressions of orfA and orfC proteins in yeast and tumor cells were checked successfully with the anti-orfA and anti-orfC rabbit serums.【Conclusion】The results showed that the polyclonal antibodies were prepared with high specificity and could be used in Western blot analysis for the expression of the target proteins in bacteria,yeast and mammalian cells.%【目的】制备大眼鰤鲈鱼真皮肿瘤病毒（WDSV）辅助基因orfA和orfC的多克隆抗体，并用其检测酵母和肿瘤细胞中目的蛋白的表达。【方法】以WDSV基因组序列为模板，PCR扩增WDSV orfA和orfC基因，分别构建其原核表达载体，转化Rosseta（DE3）感受态细胞，经IPTG诱导表达并纯化重组蛋白后，采用皮下多点注射结合耳静脉加强免疫的方法免疫新西兰白兔，制备抗orfA和抗orfC的多克隆抗体，用Western blot法检测抗体的特异性。用制备的多克隆抗体检测orfA和orfC融合蛋白在酵母和HeLa299、SPC-A-1肿瘤细胞中的表达。【结果】PCR扩增获得了WDSV orfA和orfC基因。成功构建了orfA和orfC基因的原核表达载体并进行了诱导表达，orfA和orfC重组融合蛋白以包涵体形式存在；采用皮下注射结合耳静脉加强免疫有效地提高了抗体效价，成功制备得到了兔抗orfA和兔抗orfC血清，抗体效价达1∶8 000～1∶10 000；通过Western blot法，利用上述抗血清分别检测到了原核表达的目的蛋白及酵母和肿瘤细胞中表达的目的蛋白。【结论】制备得到的抗orfA和抗orfC多克隆抗体具有很好的特异性，能够用于原核表达、酵母和肿瘤细胞中表达的目的蛋白的Western blot检测。