木尔坦棉花曲叶病毒编码4个蛋白的抗血清制备

Preparation of Antisera Against Four Proteins Encoded by Cotton Leaf Curl Multan Virus

【目的】对入侵我国的木尔坦棉花曲叶病毒(Cotton leaf curl Multan virus,CLCuMuV)编码的V1、V2、C1和C4蛋白抗血清进行制备,为CLCuMuV的检验检疫及其致病机制的研究奠定基础。【方法】利用原核表达技术对V1、V2、C1和C4蛋白进行IPTG诱导表达,western blot分析表达蛋白的特异性后,V1、V2、C1蛋白免疫新西兰大白兔,C4蛋白免疫小白鼠制备抗血清,利用western blot对抗血清的特异性进行分析。【结果】通过PCR分别扩增V1(768 bp)、V2(363 bp)、C1(1089 bp)和C4(300 bp)目的基因片段,分别连接于原核表达载体后,通过SDS-PAGE和western blot分析分别在34、16、44、13 kDa处存在与预期蛋白大小一致的条带,表明该4种蛋白均成功表达。Western blot分析发现获得的4种抗血清均具有较高的特异性。【结论】成功制备了CLCuMuV V1、V2、C1和C4蛋白的抗血清,可用于后续建立CLCuMuV的检疫方法及研究其致病机制。

【Objective】Antisera of V1,V2,C1,and C4 proteins encoded by the invasive cotton leaf curl Multan virus(CLCuMuV)were prepared for quarantine at entry ports and study on the pathogenic mechanism of the virus.【Method】Using a prokaryotic expression technology and IPTG induction,V1,V2,C1,and C4 were expressed and the specificity analyzed by western blot.Separately,New Zealand white rabbits were immunized with V1,V2,and C1,while mice with C4 to prepare the antisera.Specificity of the antisera was subsequently verified by western blot.【Result】The target gene fragments of V1(768 bp),V2(363 bp),C1(1089 bp),and C4(300 bp)were amplified by PCR and connected to the respective prokaryotic expression vectors.The SDS-PAGE and western blot showed that the 4 proteins were successfully expressed at 34,16,44,and 13 kDa locations with high specificity on the respective antiserum.【Conclusion】The antisera of CLCuMuV V1,V2,C1,and C4 were successfully prepared for quarantine inspection and study on the pathogenic mechanism of the invasive virus.