Prokaryotic Expression, Polyclonal Antibody Production And Detection Of Tobacco Curly Shoot Virus Coat Protein

Geminiviruses are single-stranded DNA plant viruses which have geminate particle morphology. Tobacco curly shoot virus (TbCSV) is a kind of momopartite geminivirus, and it belongs to genus Begomovirus and family Geminivirdae. TbCSV causes severe losses on yield of tobacco and tomato plants in Yunnan Province. In order to further understand the impact of TbCSV and begomoviruses in fields in other provinces of our country and the interaction between TbCSV and its host plants, the coat protein gene (CP) of TbCSV was cloned and prokaryotically expressed to prepare polyclonal antibody. Symptomatic samples were collected from fields and detected for virus infection by using PCRand ELISA technology based on preparation of polyclonal antibody of TbCSV CP.TbCSV CP (771 bp) was cloned with specific primers and subcloned into pGEX-6P-1, a prokaryotic expression vector. GST-CP. which had a glutathione S-transferase mark and existed as a inclusion body, was successfully expressed in Escherichia coli BL21(DE3)plus S. The concentration of GST-CP was concentrated to 1 mg/ml by PEG 20,000. The purified GST-CP protein was injected into a healthy rabbit to generate polyclonal antibody. The titer of TbCSV CP antibody was determined as 1:10,000 by the ACP-ELISA technology. Western-blot results showed that the specificity of this kind of antiserum was well enough.Seventy six field weeds samples of Malvastrum coromandelianum, collected from Panzhihua regions in Sichuan Province, detected by ELISA and PCR. Tweenty three samples were positive by ELISA using polyclonal antibody against TbCSV CP. PCR results showed that these 23 samples were infected by geminivirus whereas 4 out of these 23 samples had TbCSV infection.In this thesis, TbCSV CP was prokaryotically expressed. The field weeds samples of Malvastrum coromandelianum were detected by ELISA and PCR. Comparison of ELISA and PCR results showed that polyclonal antibody of TbCSV CP can be used to detect begomoviruses, indicating there is a close serological relationship among members of Begomovirus. Thus, this antibody can be used to make large amount of detections of begomoviruses in field. These results also suggest that begomoviruses have been found widely in Panzhihua regions of Sichuan province, therefore a great attention should be paid to its possibility of undermining further. TbCSV is sporadically distributed and not a dominant virus species in Panzhihua regions.