The Research Of Foot-and-mouth Disease Virus Receptor Integrin β6　Genes From Bovine

Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease of infecting the cattle, sheep, pigs and other cloven-hoofed animals infected with foot-and-mouth disease virus (FMDV), which cause great loss of animal husbandry. Integrin is one of the important receptors that mediated FMDV infection, whereas whether it play a decisive role and its mechanism is unclear. This article was prepared polyclonal antibody against bovine integrinβ6 subunit, a cell line stable expression of integrinβ6 subunit was established, expression of integrinβ6 subunit in different tissues was determined. The specific findings are as follows:1. Bovine integrinβ6 subunit gene prokaryotic expression, purification and its polyclonal antibody preparationTotal RNA was extracted from bovine thyroid tissue, according toβ6 sequence in GenBank, primers P1 and P2 were designed and synthesized, andβ6 cDNA amplified using RT-PCR, then subcloned into pEASY-T3 vector, and sequenced. Sequence analysis showed thatβ6 cloned gene sequences was 99% homology with the published sequence.β6 recombinant pEASY-T3 plasmid was template,β6 extracellular domain gene was amplifed with P3 and P4 primers, and then restructed into the prokaryotic expression vector pET32a and transformed into BL21 (DE3) bacteria.The plasmid were extracted from positive clones and extracellular domain ofβ6 recombinant prokaryotic expression plasmid pET32a-β6 were confirmed with Enzyme digestion, PCR and sequencing analysis. The expression the extracellular domain ofβ6 fusion protein was induced with IPTG, most expressed protein exists in inclusion bodies, and its molecular weight is about 94kD. The protein was purified with Ni-NTAbeads and the New Zealand rabbits were immunized with the fusion protein, then polyclonal antibodies againstβ6 fusion protein were collected. Using this antibody as a first antibody, theβ6 subunit expressed by a PK15 cell lines stably expressing bovineβ6 were determined. With Western Blot, the antibody was high specificity forβ6 protein.2. Bovine integrinβ6 subunit gene eukaryotic expression and stable expressing cell lines were established Primers P5 and P6 were designed and synthesized, theβ6 cDNA was amplified fromβ6 pEASY-T3 recombinant plasmid by PCR with P5 and P6 primers, its fragment digested by BglПand NotI was recovered, connected into FG9 vector to construct the eukaryotic expression vector FG9-β6, then confirmed by sequence analysis. The recombinant plasmids was transfected into 293T cell, the transient expression ofβ6 protein was determined with Western Blot. After the success of transient expression of the recombinant plasmid, FG9-β6 was transfected into FMDV non-sensitive pig kidney cell line PK15, GFP-positive cell were screening with flow cell sorting, and bovineβ6 expression was confirmed with Western Blot.3. mRNA expression of Bovine integrinβ6 subunit in different tissues was determinedReal-time quantitative PCR was established to detect bovine integrinβ6 sbunit mRNA expression. The results show thatβ6 could be expressed in cattle 10 kinds of tissues at different levels, in the kidney and the tongue at high levels, followed by heart, thyroid, tongue, spleen, lung, the toes and in liver, intestine, trachea, testis at very low expression level. High expression ofβ6 in bovine tissues is coincided with FMDV high sensitive tissues in vivo, indicating that theβ6 subunit may play an important role in FMDV natural infection.