Efficient Expression Of RGMoV Coat Protein Gene In E.coli

Ryegrass, also called Italy ryegrass, is a kind of annual or biennial plant. Ryegrass is of high quality grass and rich in nutrition, so various livestocks all like to eat the grass. Ryegrass is a kind of good succulent fodder for its rapid growth rate, strong stooling, good reproducing property and high yield. In addition, ryegrass is a common grass planted for city landscaping.Ryegrass mottle virus is a common pathogen of ryegrass which can lead to mottle and mosaic symptom and seriously affect the qulity and yield of the grass. The virus has not been reported in China so it is necessary to develop reliable and sensitive methods for the identification of the virus. Serology method is widely used in virus detection because the method is sensitive, rapid and suitable for simultaneous detection of many samples. People attempt to obtain coat protein of virus by gene engineering method for many plant viruses are hard to purification. The recombinant coat protein is used for antiserum preparation which will be applied in virus inspection.Based on published nucleotide sequence, coat protein gene of RGMoV (Ryegrass mottle virus, RGMoV) was obtained by reverse transcription polymerase chain reaction (RT-PCR) and constructed into expression vector pGEX-4T-l. And then objective protein was expressed by IPTG induction. The result of SDS-PAGE showed that successful expression of a 52kD fusion protein was achieved by induction, and furthermore it was indicated by immunoblotting that the fusion protein was expressed in the form of insoluble inclusion bodies. The raw inclusin body was washed by inclusin bodies washing buffer(containing 2% DOC) in order to remove soluable proteins which coprecipitate with inclusin bodies.The washed inclusion bodies were dissolved by 6M Glu-HCl. Then the interested protein was recovered from denatured form.Coat protein of ryegrass mottle virus was successfully obtained by prokaryotic expression system and the interested protein was primarily purified. So antiserum against the interested protein was available.