Expression, Purification And Anti-fibrotic Activity Analysis Of The Truncated Human Transforming Growth Factor Beta Type II Receptor Extracellular Domain

Objective:To construct the prokaryotic expression vector containing the extracellular domain of transforming growth factor-beta receptor type ??thT?R??,express and characterize the recombinant protein.Methods:The gene fragment of thT?R? from recombinant vector pGEX-4T1-T?R?was amplified by regular PCR with specific primers,and then cloned into the prokaryotic expression vector pET28a.The recombinant protein pET28a-thT?R?were induced with IPTG,purified with Ni²⁺-IDA affinity chromatography and characterized with SDS-PAGE and MTT assay.Expression of TGF?1?FNmRNA and FN??-SMA protein in LX-2 cells which were treated with TGF-?1 and different concentrations of recombinant protein pET28a-thT?R? were detected by real-time RT-PCR and Western bloting respectively.Results:The PCR product of thT?R? digested with two restriction enzymes?NdeI and BamHI?was cloned into the expression vector pET28a,and the sequence of the gene was confirmed by PCR,restriction endonuclease digestion and DNA sequence.The optimal expression of recombinant protein thT?R? was induced with0.8 mM IPTG at 37°C for 5 h.The high-level expressed protein in the form of inclusion bodies was refolded by dialysis refolding procedures and purified by Ni²⁺-IDA affinity chromatography,identified by SDS-PAGE.The recombinant protein thTGF-?R?,at concentrations of 150?g/mL,could significantly inhibit the proliferation of TGF-?1-induced LX-2 cells;at concentrations of 200?g/mL,could significantly decrease the expression level of TGF-?1?FNmRNA and FN??-SMA protein.Conclusions:The pET28a-thT?R? expression vector was constructed,and the production of active recombinant protein thT?R? could inhibit the proliferation of TGF-?1-induced LX-2 cells?decrease the mRNA and protein expression levels of fibrosis-related factors,which lays the foundation for exploring the effect of the recombinant protein thT?R? in vitro and in vivo.