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为了分析草莓镶脉病毒(Strawberry vein banding virus,SVBV)ORFⅥ基因的功能,采用原核表达技术获得该基因表达的重组蛋白并制备其抗血清。利用PCR技术扩增得到SVBV中国分离物的ORFⅥ基因,将此基因克隆到原核表达载体pET-SUMO上,获得重组质粒pET-SUMO-ORFⅥ。转化大肠杆菌BL21(DE3)后,经IPTG诱导与Ni2+-NTA亲和柱纯化,获得分子质量约为90 kD的重组蛋白。以纯化的重组蛋白为抗原免疫家兔制备抗血清,采用间接ELISA和Western blot方法测定抗血清的效价、反应灵敏度以及ORFⅥ基因在本氏烟中的瞬时表达。结果显示,间接ELISA法测定抗血清对重组蛋白的效价达1∶256 000,Western blot法能够检测到稀释64 000倍的重组蛋白。利用稀释2000倍的抗血清,仍能够检测出SVBV中国分离物和美国分离物ORFⅥ基因在本氏烟中瞬时表达的蛋白。
In order to analyze the function of the ORFⅥ gene of Strawberry vein banding virus (SVBV), the recombinant protein expressed by this gene was obtained by prokaryotic expression and the antiserum was prepared. The ORFVI gene of SVBV Chinese isolate was amplified by PCR and cloned into the prokaryotic expression vector pET-SUMO to obtain the recombinant plasmid pET-SUMO-ORF Ⅵ. After transformed into E. coli BL21 (DE3), it was purified with NiT2-NTA affinity column by IPTG induction to obtain a recombinant protein with a molecular mass of about 90 kD. The purified recombinant protein was used as an antigen to immunize rabbits to prepare antiserum. The antiserum titer, reaction sensitivity and transient expression of ORFⅥ gene in N. benthamiana were determined by indirect ELISA and Western blot. The results showed that the titer of recombinant protein was 1: 256 000 by indirect ELISA, and 64 000-fold recombinant protein was detected by Western blot. Using a 2000-fold diluted antiserum, one can still detect proteins transiently expressed in N. benthamiana from SVBV China isolate and U.S. isolates ORFⅥ gene.