Establishment Of A Stable αvβ3-expressing Cell Line And Evaluation Of Its Susceptibility To Foot-and-mouth Disease Virus

Foot-and-mouth disease virus(FMDV), the prototype member of the Aphthovirus genus within the picornaviridae family, is a single-stranded, positive-sense RNA genome virus, which affects many stock cloven-hoofed animals, including cattle, buffalo, swine, goat, sheep and other wildlifes. Virus identification receptor is the first step of virus infection, determining the cell type and host tropism. FMDV binds RGD motif of integrin receptor(αvβ1, αvβ3, αvβ6 and αvβ8) via the clathrin-mediated endocytosis pathway; FMDV also enters to the cells using the caveola-mediated endocytosis pathway by VP3056 R binding heparan sulfate. In addition to mediate virus infection, integrin receptors also can stimulate signal transduction and play other roles, but we understand relatively little about this function and mechanisms. Therefore, in order to reveal the roles of a single integrin receptor in virus infection and signal transduction, we established cell line which lays a foundation for further study. In this study, we clone the genes of αvβ3 integrin receptors, establish a CHO-677 cell line stably expressing αvβ3, and evaluate the susceptibility to FMDV. As follows:(1) Cloning αv and β3 genes: Genomic RNA of integrin αv and β3 subunit genes was extracted from the tongue or lung tissues of suckling mice. According to the published gene sequences in GenBank, we designed two pairs of specific primers, respectively. Murine αv and β3 subunit genes were amplified using RT-PCR, and then connected with the pGEM-T vector. The recombinant plasmids were sequenced by GeneWiz Inc. The results showed that the αv PCR fragment of 3135 nucleotides and the β3 PCR fragment of 2364 nucleotides. The suckling mouse αv sequence was highly homologous to the human and swine, while β3 sequence was highly homologous to the sheep and cattle.(2) Expressing protein and preparing polyclonal antibody: Part of extracellular domain of αv and β3 subunit genes were cloned into the prokaryotic expression vector pET-30 a. The recombinant expression plasmids were constructed and transformed into BL21(DE3). The recombinant proteins were expressed after IPTG induction and detected by SDS-PAGE. The New Zealand rabbits were immunized with the purified fusion protein to prepare polyclonal antibodys, which were identified by Western blot. SDS-PAGE demonstrated that the fusion protein was expressed efficiently as inclusion body with the expressed molecular weight of 40 kDa and 40 kDa, separately. Western blot assay showed that the recombinant protein has the good antigenicity and specificity.(3) Establishment of cell line expressing the murine αvβ3: A stable CHO-677 cell line expressing the murine αvβ3 heterodimer was established using a highly efficient lentviral-mediated gene transfer technique. Integrin subunits αv and β3 were detected at the gene and protein levels with PCR and IFA, respectively, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably.(4) Evaluation of susceptibility to FMDV: A plaque-forming assay, TCID50, Q-RT-PCR, and IFA were used to detect the replication levels of FMDV in the CHO-677-αvβ3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNAs and proteins, compared with those in CHO-677 cells. These data suggested that we successfully established a stable αvβ3-receptor-expressing cell line with increased susceptibility to FMDV.