Cloning And Overexpression Of Goat Toll-like Receptor 2 Gene

Toll-like receptors (TLRs) were identified as pattern–recognition receptors, which activated the innate and acquired immune systems. Among the TLRs family members, TLR2 was one of the most ligands identified receptor. TLR2 attracted much attention, because it played an important role in many infections caused by pathogenic microorganisms. At present, there is no relevant report on goat TLR2 in China.In this study, goat Toll-like receptor 2 gene was cloned by RT-PCR from goat spleen tissue. After identified by sequencing, goat TLR2 was connected to the pEGFP-N1 vector and constructed green fluorescent protein fusion vector pEGFP-TLR2-N1. Expression of green fluorescent fusion protein was observed under fluorescence microscope after the fusion vector transfected into 293T cells. 3S-Loxp-TLR2 recombinant vector was transfected into goat fibroblasts and positive clone cells were purified by G418. The DNA of the positive cells was extracted for PCR. Expression of TLR2 was detected by real-time quantitative PCR.The goat TLR2 gene was cloned and submitted to GenBank, serial number: GU984768.1. The sequence analysis showed that the ORF of TLR2 gene was 2355bp, encoding 784 amino acids. The goat TLR2 was alignmented with other published sequences of TLR2s, the results showed that the similarity of goat TLR2 to theirs of sheep, cattle and Indian nilgai were much higher than pigs, horses and dogs; the lowest was that of chicken. The structural prediction demonstrated that the goat TLR2 was a typical typeⅠtransmembrane protein containing extracellular, transmembrane and intracellular region.The pEGFP-TLR2-N1 fusion vector was transfected into 293T cells using calcium phosphate method, and the results showed that green fluorescent fusion protein was localized mainly in the cell membrane. Through 3S-Loxp-TLR2 recombinant vector was transfected into goat fibroblasts and positive clone cells were purified by G418, the goat TLR2 gene was overexpressed in the positive clone cells. There was no significant difference between the transfected goat fibroblasts cells on cell condition and that in control. This work is helpful to further study the functional mechanism of goat TLR2 and to product transgenic goat using somatic cell cloning.