Bacteria expression and antisera preparation of non-structural protein encoded by triple gene block of Cowpea mild mottle virus

• Main Authors: Chen-Chung Hsu, 徐晨中
• Other Authors: Chin-An Chang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/78840917266319423284

碩士 === 朝陽科技大學 === 應用化學系生化科技碩士班 === 101 === Common bean (Phaseolus vulgaris) is one of the important legume crops in Taiwan. Its wide adaptability makes it suitable for growing form south to north in this island. Its annual cultivated area and production are over 2,000 hectares and about 12 tons, respectively. Common bean is acrop prone to be infected by various viruses which have severely interfered its production and quality worldwide. However, the Cucumber mosaic virus(CMV) is the only virus being reported on common bean so far in Taiwan. Common bean plants with symptoms of mosaic, leaf shrinkage and dwarf had been collected form nantou County in 2008. Cowpea mild mottle virus(CpMMV), a Carlavirus, has been identified as the causal agent. CpMMV was first found on cowpea in east India and Ghana, and spreaded to Africa, the Middle East and Asia later on. It causes symptoms of mosaic, chlorotic spots, and leaf distortion on cowpea, and can be transmitted by whitefly (Bermisia tabaci) in a non-persistent manner. As a Carlavirus, the genome of CpMMV contains an overlapping gene region called the triple gene block, (TGB), which encodes three functional proteins mediated the virus movement in the early stage of infection in planta. The objectives of this thesis are to produce antisera against individual functional protein of CpMMV TGB and to establish possible detection methods for detecting CpMMV by using antisera produced in serological tests such as enzyme-linked immune-sorbent assay (EELISA) and western blotting test. For the cloning of full-length TGB, corresponding primers (TGB-up1/TGB-dw1) were designed based on the full-length sequence of CpMMV (HQ184471) available on NCBI GenBank database. A cDNA fragment of about 1200 bp was obtained after reverse-transcription polymerase chain reaction (RT-PCR) and was cloned into pGEM-T easy vector. Sequence analysis has confirmed the cloned cDNA harbored all three ORFs, i.e. ORFs 2, 3, and 4, of TGB. The ORF2 consists of 696 bp whereas ORFs 3 and 4 contain 321 bp and 207 bp respectively. Individual ORFs of TGB were further cloned into bacterial expression vector pET-28 (b+) separately after PCR amplification with primer sets of (ORF2-up/ORF2-dw; , ORF3-up /ORF3-dw; , and ORF4-up /ORF4-dw), accordingly. Each expression constructs were then transformed individually into E. scherichia coli (strain BL21) for the over-expression of each recombinant proteins. The over-expressed proteins of 26 kDa, 12 kDa and 8 kDa, corresponding to the products of ORFs 2, 3 and4, respectively. , can be visualized on SDS-PAGE. Bacterial expressed ORF2 protein was purified by using preparative gel electrophoresis. As the protein products of ORF3 and ORF4 were too small to be purified as ORF2 protein, the metal ion affinity chromatography (or immobilized metal affinity chromatography, IMAC) was used as an alternative. The purified proteins were then used as antigen for the production of specific polyclonal antibodies in rabbit. The obtain antibodies were used to detect the CpMMV infection. CpMMV-infected leaves of common bean were collected three, six and nine days post inoculation (dpi). The presence of the protein products of CpMMV ORFs 2, 3, and 4 in inoculated and systemic leaves were tested by ELISA and Western blot analyses. The ORF2 protein of 26 kDa can be detected in the inoculated and systemic cowpea leaves of 3-9 dpi was not detectable in infected leaves of 12 dpi, in ELISA and Western blot tests. However, the protein of CpMMV ORF3 only can be detected in the leaves of 3 dpi in immunoblots while the ORF4 protein was detectable in the leaves of 3 dpi with both ELISA tests and iimmunoblots. These result indicated that the CpMMV TGB proteins accumulated only in the early stage of infection, which is in accord with those data showed in earlier documentations. In brief, the results of this thesis showed, the prepared antisera ahainst CpMMV TGB are suitable for applying to detect target proteins in vitro and in vivo.