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Expression, purification and characterization of the Lily symptomless virus coat protein from Lanzhou Isolate.

ABSTRACT: BACKGROUND: Lily symptomless virus (LSV) is widespread in many countries where lily are grown or planted, and causes severe economic losses in terms of quantity and quality of flower and bulb production. To study the structure-function relationship of coat protein (CP) of LSV, to investigate antigenic relationships between coat protein subunits or intact virons, and to prepare specific antibodies against LSV, substantial amounts of CP protein are needed. RESULTS: Thus, full-length cDNA of LSV coat protein was synthesized and amplified by RT-PCR from RNA isolated from LSV Lanzhou isolate. The extended 33.6 kDa CP was cloned and expressed prokaryoticly and then purified by Ni-ion affinity chromatography. Its identity and antigenicity of recombinant CP were identified on Western-blotting by using the prepared anti-LSV antibodies. CONCLUSIONS: The results indicate that fusion CP maintains its native antigenicity and specificity, providing a good source of antigen in preparation of LSV related antibodies. Detailed structural analysis of a pure recombinant CP should allow a better understanding of its role in cell attachment and LSV tropism. This investigation to LSV should provide some specific antibodies and aid to development a detection system for LSV diagnostics and epidemiologic surveys.

SUBMITTER: Wang R

PROVIDER: S-EPMC2828416 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Expression, purification and characterization of the Lily symptomless virus coat protein from Lanzhou Isolate.

Wang Ruoyu R Wang Guangpeng G Zhao Qi Q Zhang Yu Y An Lizhe L Wang Yun Y

Virology journal 20100210

<h4>Background</h4>Lily symptomless virus (LSV) is widespread in many countries where lily are grown or planted, and causes severe economic losses in terms of quantity and quality of flower and bulb production. To study the structure-function relationship of coat protein (CP) of LSV, to investigate antigenic relationships between coat protein subunits or intact virons, and to prepare specific antibodies against LSV, substantial amounts of CP protein are needed.<h4>Results</h4>Thus, full-length c ...[more]

PMID: 20144245

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Project description:BackgroundGarlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors.ResultsIn this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR.ConclusionsThe expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

Project description:Low temperature induces changes in plants at physiological and molecular levels, thus affecting growth and development. The Lanzhou lily (Lilium davidii, var. unicolor) is an important medicinal plant with high economic value. However, the molecular mechanisms underlying its photosynthetic and antioxidation responses to low temperature still remain poorly understood. This study subjected the Lanzhou lily to the two temperatures of 20°C (control) and 4°C (low temperature) for 24 h. Physiological parameters related to membrane integrity, photosynthesis, antioxidant system, and differentially expressed genes were investigated. Compared with control, low temperature increased the relative electrical conductivity by 43.2%, while it decreased net photosynthesis rate, ratio of variable to maximal fluorescence, and catalase activity by 47.3%, 10.1%, and 11.1%, respectively. In addition, low temperature significantly increased the content of soluble protein, soluble sugar, and proline, as well as the activity of superoxide dismutase and peroxidase. Comparative transcriptome profiling showed that a total of 238,109 differentially expressed genes were detected. Among these, 3,566 were significantly upregulated while 2,982 were significantly downregulated in response to low temperature. Gene Ontology enrichment analysis indicated that in response to low temperature, the mostly significantly enriched differentially expressed genes were mainly involved in phosphorylation, membrane and protein kinase activity, as well as photosynthesis, light harvesting, light reaction, and alpha,alpha-trehalose-phosphate synthase activity. Kyoto Encyclopedia of Genes and Genomes enrichment analysis also indicated that the most significantly enriched pathways involved ribosome biogenesis in eukaryotes, phenylalanine metabolism, circadian rhythm, porphyrin and chlorophyll metabolism, photosynthesis of antenna proteins, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, the expression patterns of 10 randomly selected differentially expressed genes confirmed the RNA-Seq results. These results expand the understanding of the physiological and molecular mechanisms underlying the response of the Lanzhou lily to low temperature stress.

Dataset Information

Expression, purification and characterization of the Lily symptomless virus coat protein from Lanzhou Isolate.

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Expression, purification and characterization of the Lily symptomless virus coat protein from Lanzhou Isolate.

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