Expression Of Tobacco Vein Banding Mosaic Virus Coat Protein In E.coli And Preparation Of Immunochromatography Test Strip

The tobacco vein banding mosaic virus disease, induced by Tobacco vein banding mosaic virus (TVBMV) of Potyvirus, happens in many main areas planted tobaccos in China. And recently it spreads and the loss caused by it is trending up. Detecting this disease correctly and in time is the important precondition of controlling it efficiently. Besides TVBMV, tobaccos infected by Potato virus Y (PVY), Tobacco vein mottling virus (TVMV) and some other members of potyvirus also present the symptom of vein banding. And what's more, complex infection is serious, so it is difficult to detect TVBMV according to symptom. A variety of methods such as ELISA and RT-PCR have been utilized to determine TVBMV. These methods, however, are instrument dependent, time consuming and inconvenient. To develop a rapid, specific and easy assay for testing TVBMV, this article prepared the test strip of TVBMV. The results are as follows:The coat protein (CP) gene of Tobacco Vein Banding Mosaic Virus (TVBMV) from Shaanxi province was obtained by means of RT-PCR. Sequence analysis showed that TVBMV CP gene was 816 nucleotides in length, which encodes the coat protein of 271 amino acids. It shared the sequence homology of 90.28 %~98.15 % nucleotide acid and 97.05 %~100 % amino acids with 9 strains of TVBMV CP registered in GeneBank., and shared highest homology with Mengyin of Shandong.The CP gene was cloned into pMD18-T Simple Vector, which were cleaved with restrict endonuclease BamH I/Not I, then inserted into PET30a. We constructed the expression vector of pET30-TVBMV CP which was transformed into BL21 host strain of E.coli and induced by IPTG. The expression product was used as antigen to immunize the rabbit and the special antiserum was prepared. The TVBMV CP highly expressed in BL21 and the molecular size of expression product was about 37 kD. The antiserum gave a titer of 1/6400 with high specificity to TVBMV. The result of western-blot revealed that the prepared antiserum can be used to detect the tobaccos infected by TVBMV.Based on the principle of colloidal gold labeling technique and immunochromatography assay, the coupling product from colloidal gold and rabbit polyclonal antibody against TVBMV was sprinkled on the glass fiber film; rabbit polyclonal antibody and sheep anti-rabbit IgG were sprinkled on the test line and control line of nitrocellulose membrane, colloidal gold strip was executed to detect TVBMV quickly. Extract from infected tobacco leaf was detected to a dilution of 10-2 and the test time was only 3~10 minutes. Non-specific reaction was not observed for testing other four main viruses infecting tobaccos. And the test strips could be stored at 4℃for more than one month. The test results in the field by strips accorded with those by RT-PCR.