Prokarkaryotic Expression Of Three Kinds Of Garli Viruses Liuan Isolates CP Gene And Test Kit Manufacture

The virus diseases of garlic are widespread throughout the world, and cause severe loss of yield and poor quality. With trade among countries increasing, garlic virus diseases spread faster in recent years and great loss has taken place. The plant viruses infectting garlic include Shallot latent virus(SLV, member of Carlavirus), Garlic common latent virus,(GarCLV, member of Carlavirus), Scallion visus x (ScaVX, member of Potxvirus), Leek yellow stripe virus (LYSV, member of Potyvirus), Onion yellow dwarf virus (OYDV, member of Potyvirus) and several members of Allexivirus(Garlic virus A, Gary-A; Garlic virus B, Gary-B; Garlic virus C, Gary-C; Garlic virus D, Gary-D; Garlic virus E, Gary-E; Garlic virus X, Gary-X).Garlic fresh leaves showing typical mosaic and mottle symptoms were collected at Jinan Area in Liuan City. Total RNA was extracted and total cDNA was synthesised. Specific primers on the basis of GarVX, LYSV and OYDV sequencewere designed respectively to amplify their CP genes.GarVX-CP, LYSV-CP and OYDV-CP genes were inserted into pSBET vector and expressed in Escherichia coli BL21 (DE3) plys S strain. The object protein was purified by 12%SDS-PAGE firstly and subsequently 5%-20%gradient SDS-PAGE. The antiserum against the CPs was raised in mice and its specificity was confirmed by Western blot analysis. IgG against GarVX-CP, LYSV-CP and OYDV-CP was purified by ammonium sulfate sedimentation firstly and subsequently by Protein A-Red Sepharose affinity chromatography. The value of three kinds of IgG was beween 1:3800-1:5000. Indirect ELISA was used to detect the occurrence of three kinds of garlic viruses. It was indicated that they all widely spread and commonly existed. The IgG prepared in this study could be suitable for GarVX, LYSV and OYDV detection.