Cloning And Expression Porcine Integrin Genes In Prokaryotic And Eukaryotic Expression Systems

Foot-and-mouth disease (FMD) is a highly contagious and economically significant disease of cattle, pigs, sheep, and wild cloven-hoofed species, which is classified by Office International des Epizooties (OIE; World Organisation for Animal Health) as the first one of OIE List A diseases. Foot-and-mouth disease virus (FMDV) belongs to the aphthovirus genus of the Picornaviridae family and seven distinct serotypes of FMDV, have been defined, namely types O, A, C, Southern African Territories (SAT) 1, SAT2, SAT3 and Asia1. It has been demonstrated that there are at least five receptors for FMDV, including integrinαvβ1,αvβ3,αvβ6,αvβ8 and heparan sulfate.In the present study, 3 pairs of primers was designed based on the integrin gene sequences of several other animnals available in GenBank. Theαv,β1 andβ6 genes are amplified by RT-PCR from RNA purified from the tongue and lung of recovered pig infected experimentally with OA/58 FMDV, and cloned into pGEM-T Easy vector to construct the recombinant plasmid pGEM-αv, pGEM-β1, pGEM-β6 for sequencing. The result showed that the new genes we cloned have high similarity with the corresponding gene of bovine in GenBank in nucleotide and amino acid sequence, with similarity above 90%.Prokaryotic expression primers were designed according to the sequence of the genes, and the pGEM-αv, pGEM-β1, pGEM-β6 recombinant plasmid used as template in the PCR reaction to amplify the fragments encoding the ligand-binding-domains (LBDs) ofαv,β1 andβ6 subunit. The amplified product ofαv was ligated to the pPROExHTb, and that ofβ1 andβ6 were ligated to the pGEX-4T-1 vector, then transformed into BL21(DE3) host cells. Expression of the recombinant plasmid was induced with IPTG, and resulted in a high level of protein expression, most of which were inclusion body. SDS-PAGE experiments revealed that molecular weight ofαvLBD is about 40000, and 42000 for GST-β1LBD and GST-β6LBD respectively. After inclusion body prepared and fusion protein purified, New Zealand rabbits were immunized to prepare polyclonal antibody againstαvLBD, GST-β1LBD and GST-β6LBD. ELISA and Western Blot demonstrated that the titers of the polyclonal antibody were all above 1: 12800 and had obvious specificity.Eukaryotic expression primers forβ6 gene were designed according to the sequences flanking pGEM-β6 Open Reading Frame (ORF) and theβ6 gene was amplified, using pGEM-β6 as temple. The PCR product was ligated with the vector pCDNA3.1/(+) to construct expression vector pCDNA3.1(+)/β6, and then transfected into Human Colon Carcinoma Cell line(SW480). Indirect immunofluorescence indicated that the pCDNA3.1(+)/β6 transfected cells SW480expressedαvβ6 whereas SW480 and pCDNA3.1/(+) transfected cells did not expressβ6.