草莓镶脉病毒ORF Ⅵ基因的原核表达与抗血清制备

Prokaryotic expression and antiserum preparation of ORF Ⅵ encoded by Strawberry vein banding virus

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 gene ORF Ⅵ of Strawberry vein banding virus （SVBV）, recombinant protein expressed by the gene was obtained using prokaryotic expression technique. Gene ORF Ⅵ of Chinese isolate of SVBV was amplified by PCR. This gene was cloned into prokaryote expression vector pET-SUMO. After the recombinant plasmid pET-SUMO-ORF Ⅵ was transformed into Escherichia coli BL21 （DE3）, the recombinant protein with an approximate molecular weight of 90 kD was obtained with IPTG induction and Ni2＋-NTA affinity column purification. Antiserum was prepared using pure recombinant protein as antigen to immunize rabbits. Titer and reaction sensitivity of the antiserum, and the transient expression of gene ORF Ⅵ in Nicotiana benthamiana was assayed by indirect-ELISA and Western blot method. The results indicated that the titer of antiserum to recombinant protein reached 1：256 000 by indirect-ELISA detection, and the recombinant protein at a dilution of 1：64 000 could be detected with Western blot method. Moreover, the transient expression protein of gene ORF Ⅵ of Chinese isolate and American isolate of SVBV in N.benthamiana could be detected with indirect-ELISA using 2 000 times diluted antiserum.