Preparation Of Antibodies Against Coat Proteins Of Two Tobacco Virus And Effects Of Phenoazine Derivatives On Virus Replication

Tobacco virus diseases are widely distributed and harmful,which is one of the main restricting factors affecting tobacco yield and quality.More than 30 viral pathogens have been reported on tobacco in China,causing persistent harm.Among them,Potato virus Y(PVY)has been increasing from the secondary pathogen to the main pathogen since the 1990 s,and has been harmful to the present.In recent years,Tomato chlorosis virus(To CV)has been reported to infect tobacco.In view of the lack of effective control agents for plant viral diseases,early and rapid diagnosis of viral diseases is particularly important.In this paper,the detection technology of PVY and To CV tobacco viruses was studied by cloning their coat protein genes,using prokaryotic expression system to express and purify their coat proteins,immunizing animals to prepare polyclonal antibodies,in order to establish the serological rapid detection technology of these two viruses.Meanwhile,in view of the fact that N-(1’-phenazine-ethyl)-phenazine-1-amide(SQXA-12(3p))can inhibit the replication of Avian infectious bronchitis virus(IBV)at the cellular level,the inhibitory effect of six derivatives of phenazine on plant virus was tested.The specific results are as follows:According to the sequence of PVYN and To CV genome in NCBI,primers were designed to amplify their CP gene,and the CP gene was cloned by RT-PCR,and ligated or recombined into intermediate plasmid p MD19-T or p DONR221.The target gene was transferred into prokaryotic expression vector p ET28 a or p DEST17 by enzyme digestion identification or recombinant method,and the recombinant plasmid PET28A-PVY CP and p DEST17-To CV CP were obtained.The recombinant plasmid was transformed into escherichia coli BL21 expressing strain and prokaryotic expression was carried out.Sds-page electrophoresis showed that PVY CP(48 k Da)and To CV CP(31.30 k Da)were successfully expressed.The two target proteins were purified by Ni-NTA chromatography and immunized with rabbits as antigens to obtain polyclonal antibodies against the two viral CP.Indirect enzyme linked immunosorbent assay(ELISA)showed that the antibody titers of PVY CP and To CV CP were1:12,800 and 1:12,800,respectively.Western Blot assay showed that the antiserum was 50 ng and 50 ng,respectively.Finally,Western Blot was used to detect the sensitivities of the two antiserums to the infected leaves,and the results showed that the sensitivities were 1mg/m L and 0.1mg/m L,respectively.On this basis,field sampling was used to conduct indirect ELISA test,and the results showed that the two antiserums could detect the corresponding viruses in tomato,tobacco and other hosts,respectively.Therefore,the antisera prepared for PVY CP and To CV CP can be used for field detection of these two viruses.It is well known that plant viral diseases lack effective control agents.Our group previously found that N-(1’-phenazine-ethyl)-phenazine-1-amide(SQXA-12(3p)),a phenazine derivatives,could inhibit IBV replication at the cellular le vel.Based on this,with PVY as the test object,Reagent spraying,real-time quan titative RT-PCR and Western were performed six phenazine derivatives,N-(2-flu orophenyl)-n-methylphenazine-1-formamide(3d),N-(2-chloropyridine-4-l)phenazine-1-formamide(3g),N-(2-fluorobenzyl)phenazine-1-formamide(3m),N-(3-fluorobenz yl)phenazine-1-formamide(3n),SQXA-12(3p),were detected by Blot analysis Inhibitory effects,N-(2-aminoethyl)phenazine-1-formamide(3q)on plant viruses.Results showed that two kinds of administer mode,namely the virus before s praying and spraying after inoculation,phenazine-1-carboxylic acid(PCA)an d 6 kinds of derivatives and potato Y virus replication of all have different de grees of inhibition,inhibiting effect from strong to weak,in order: 3n,3d,3g,SQXA-12(3p),3m,3q,but its inhibition is not controlled medicine building drug effect is remarkable.