茉莉H病毒外壳蛋白基因的克隆、原核表达及抗血清制备

Cloning,prokaryotic expression and antiserum preparation of coat protein gene of jasmine virus H

茉莉H病毒(jasmine virus H,JaVH)是近年来引起茉莉病毒病的一种新病毒,Zhuo等[1]在我国福建黄化嵌纹的茉莉上首次发现并报道,随后Dey等[2]在美国夏威夷和华盛顿也发现了JaVH,并认为其是引起茉莉病症不可或缺的致病因子之一。

Based on the sequence of jasmine virus H,the specific primer pairs were designed to amplify the cDNA of coat protein(CP)by PCR using plasmid from previous study as template.The PCR fragment was then cloned into the prokaryotic expression vector pET-28 a(+).Subsequently,the recombinant plasmid pET-JaCP was transformed into Escherichia coli JM109.The positive clones were then screened and sequenced.The resulting recombinant plasmid was prepared and transformed into E.coli Rosetta(DE3),which designed to enhance the expression of exogenous proteins.The CP fused with His-tag was over-expressed with IPTG induction and purified using Ni2+-NTA Agarose affinity chromatography.New Zealand white rabbits were immunized with the purified protein for specific antibody.The ELISA result of JaVH infected Nicotiana benthamiana indicated that the 1000 times dilution of prepared antiserum is best for sample detection in the three tested dilution.Western blot analysis showed that 1000 times diluted antiserum could be used to specifically detect jasmine samples carrying JaVH.