Gene Cloning, Expression And Antibody Preparation Of Potato Virus Y HC-Pro And Sugarcane Mosaic Virus CP

Serological methods have been proved to be sensitive and specific indetecting and identifying viruses. They also played great roles in studyinglocalization of viral proteins, functions of viral genes, transmissionmechanism, and virus-host interactions. However, there were severalpotential disadvantages for serological studies. For example, some virusesexisted in plants at very low concentration and were difficult to be purified.Some antisera contained non-specific antibodies to host constituents.Antibody raised against viral protein expressed in E.coli might partly solvethese problems. The main results of this study read as follows: 1. The HC-Pro genes of PVYN and PVYO were amplified by RT-PCR and their complete nucleotide sequences were determined. The results showed that they all composed of 1368 nt, each encoded a protein of 456 amino acids. Sequence comparison showed these two genes had an identity of 99.71% at nucleotide level and 99.56% at amino acid level. They share higher homology with other strains of PVY than with other viruses of Potyvirus. 2. The HC-Pro gene of PVYN was cloned into expression vector pET22b(+)and transferred into E.coli BL21(DE3). SDS-PAGE showed it could be expressed to high level when induced with IPTG, the molecular weight of the fusion protein is ca.53kD. The 53kD protein was purified from SDS-PAGE and the antiserum against the protein was raised in rabbit. 3. RT-PCR and specific primers were used to amplify the gene encoding the coat protein of Tai'an isolate of Sugarcane Mosaic virus (SCMV-TA). This sequence was cloned into pUC18 to transform E.coli DH5α. Sequence analysis showed that the CP gene of SCMV-TA included 939bp, encoding 313 amino acids. Sequence comparisons showed that it shared higher homology with isolates of Sugarcane mosaic virus than that of other viruses, indicating that the pathogen of this disease on maize in Tai'an was Sugarcane mosaic virus.4. The coat protein gene of SCMV-TA was cloned into expression vector, pET22b(+), and transferred into E. coli, BL21(DE3). It can be expressed as a fusion protein to high level(about 15% of the total protein)when induced with IPTG.. The molecular weight of the fusion protein is ca.38kD. With the protein expressed in E.coli, we prepared antiserum with high titer and specificity. The results of Western blotting confirmed the fact that the SCMV-TA CP was expressed correctly.