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In Vitro Expression and Tissue Profile of Sheep Integrin αvβ6 as FMDV Receptor

Author: XueShuang
Tutor: ChangHuiZuo；FuXiaoPing
School: Gansu Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Foot-and-mouth disease virus receptor integrinαvβ6 prokaryotic expression cell line tissue profile
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 30
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Abstract


Foot-and-mouth disease (FMD) is an acute, febricity and highly contagious disease caused by foot-and-mouth disease virus (FMDV), which is classified by Office International des Epizooties (OIE; World Organisation for Animal Health) as the first one of OIE List A diseases. It has been demonstrated that there are at least five receptors for FMDV, including integrinαvβ3,αvβ6,αvβ1,αvβ8 and heparan sulfate(HS). Integrinαvβ6 serves as the major receptor that determines the tropism of FMDV for the epithelia normally targeted by this virus.The fragment coding sheep integrinβ6 ligand-binding domain was amplified by PCR from the recombinant plasmid pGEM-β6. After doubly digested with BamHⅠand XhoⅠ, theβ6LBD fragment was subcloned into prokaryotic expression vector pPROEXTMHTb which was doubly digested with same enzymes. The recombinant expression plasmid pPRO/β6LBD was constructed and transformed into E.coli BL21(DE3) and induced by IPTG. The expression of fusion protein was detected by SDS-PAGE and purified by Ni-NTA His.Bind resins. The expressed product was identified by ELISA and Western-Blot assay. SDS-PAGE demonstrated that the fusion protein was expressed efficiently as inclusion body in E.coli with the expected molecular weight of 33KDa. ELISA and Western-Blot assays showed that the recombinant protein has the good antigenicity and specificity, which will lay a foundation for further research on distribution and the role of the ovine integrinβ6 subunit in FMDV infection.Eukaryotic expression primers forβ6 ligand-binding domain were designed and theβ6 gene was amplified, using pGEM-β6 as temple. The PCR product was ligated with the vector pcDNA3.1(+) to construct eucaryon expression vector pcDNA3.1(+)/β6S, and then transfected into Human Colon Carcinoma Cell line(SW480). The expression of integrinαvβ6 was detected by indirect immunofluorescence assay(IFA) and Western-Blot assay. IFA indicated that green fluorescence was appeared in cell membrane and cytoplasm of the pcDNA3.1(+)/β6S transfected cells, whereas, it can not be observed in SW480 and pcDNA3.1(+) transfected cells. The result of Western-Blot showed that the recombinant protein was expressed and could be recognized by the monoclonal antibody against porcine integrinβ6 subunit LBD.The integrin receptors of FMDV have been studied extensively in cell culture. However, the roles of the various integrins in determining the tissue tropism and pathogenesis of FMDV have not yet to be established. Here, we present analyses, using indirect immunohistochemistry assay (IHA), immuno?uorescence confocal microscopy and Real-time RT-PCR, ofαvβ6 expression within the different tissues that are normally targeted by FMDV in sheep. The results showed that idio-positive reaction was appeared in cell membrane and cytoplasm of tongue, nose, lip and so on,αvβ6 was mainly expressed on the surfaces of the epithelial cells at the sites where FMDV is known to replicate at a high level during a natural infection. Results of the Real-time RT-PCR study showed that theαv,β1 mRNA were transcripted at high or low level in the different tissues. In constrast, theβ6 mRNA is only restricted in the partial tissues, and at high level in the salivary gland, heart, lung and nasal epithelium, and not detected in the mandibular lymph node, liver and speen. These data suggest that integrinαvβ6 serves as the major receptor that determines virus tropism, andαvβ6 is expressed constitutively at levels sufficient to allow initiation of infection, which will lay a foundation for further understanding of the roles of integrinαvβ6 in FMDV infection.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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