Cloning Foot-and-Mouth Disease Virus Receptor Gene And Establishment Of CHO Cell Stably Transfecting Porcine Receptor β1、β3Subunit Gene

Foot-and-mouth disease virus (FMDV) is the aetiological agent responsible for foot-and-mouth disease, and is an acute，febrile，highly contagion viral disease of cloven-hoofed animaland wildlife，mostly cattle，swine，sheep and goats，leading to severe economic losses due toreduction in livestock production and restriction of trade on animal and animal products. FMDV isthe type species of the Aphthovirus genus of the Picornaviridae family. Seven serotypes (A, O, C,Asia1, and SAT1, SAT2, and SAT3) have been identified serologically, and multiple subtypesoccur within each serotype. Foot-and-mouth disease virus(FMDV)initiates infection by attachingto integrin receptors through a long surface loop, the G-H loop of VP1, which contains aconserved Arg-Gly-Asp(RGD)motif that is characteristic of the ligands of several members of theintegrin family. It has been demonstrated that four members of the integrins, αvβ1、αvβ3、αvβ6and αvβ8and heparan sulfate can serve as receptors for FMDV in cell culture.In the present study，several pairs of primers was designed based on the integrin genesequences of bovine、sheep and porcine available in GenBank. The total RNA purified from thetongue、lung and trachea of the porcine and bovine. The porcine integrin gene αv、β1、β3and β6genes,and the bovine integrin gene αv、β1、β3、β6and β8genes are amplified by PCR fromcDNA and cloned into pMD18-T vector to construct the recombinant plasmid for sequenced andanalyzed. The result showed that the porcine intergin have the most homologous with thecorresponding gene of bovine and sheep，the nucleotide sequence similarity of integrin was above93%, and the amino acid sequence similarity of integrin was above94%. and have the lowesthomologous with the corresponding gene of chicken, the nucleotide sequence similarity of integrinwas below75%, and the amino acid sequence similarity of integrin was below80%. The bovineintergin have the most homologous with the corresponding gene of sheep, the nucleotide sequencesimilarity of integrin was above97%, and the amino acid sequence similarity of integrin was above98%, and have the lowest homologous with the corresponding gene of chicken, the nucleotidesequence similarity of integrin was below74%, and the amino acid sequence similarity of integrinwas below80%. The integrins is relatively conservative in the animal species, especially in adeeply conservative between cloven-hoofed animal.Eukaryotic expression primers for the porcine integrin gene β1and β3gene were designedaccording to the sequences flanking pMD18T-β1and pMD18T-β3Open Reading Fragment (ORF)and the β1and β3gene was amplified, using pMD18T-β1and pMD18T-β3as temple. The PCRproduct was ligated with the Eukaryotic expression vector pCDNA3.1/(+) to constructrecombinant expression plasmid pCDNA3.1/(+)-β1and pCDNA3.1/(+)-β3, and then transfectedinto Chinese hamster ovary（CHO）by the Lipofectamine2000,respectively. The positive cellclones were screened with G418. In order to examine if the gene have integrated into genome ofthe cells, the genomic DNA of cell clones were used for PCR amplification. The indirect immunofluorescence is launched by the rabbit anti-human integrin β1and β3polycloneantibody,respectively,and the Western-blotting is launched by the mouse anti-human integrinαvβ3monoclonal antibody. The result indicated that the porcine integrin β1and β3protein isstably expressing CHO cells line.