| Objective: Construct prokaryotic expression vectors p ET-21a-t TβRII-6 and p ET-21a-t TβRII-G with cloning 2 receptors of truncated human transforming growth factor,t TβRII-6 and t TβRII-G.Optimization of protein expression conditions and take bioactivity analysis for or the next step in the preparation of Chinese preparation laid the foundation.Methods: Extract the RNA from human blood and transform to c DNA by reverse transcription.Target gene with t TβRII is by regular PCR amplification,insert it into the p ET-21 a of prokaryotic expression vector and name the recombinant plasmid as p ET-21a-t TβRII-G and p ET-21a-t TβRII-6.After the amplification of PCR,double restriction enzyme cutting and DNA sequecing,transport and induce the constructed p ET-21a-t TβRII-G and p ET-21a-t TβRII-6 expression in Rossta of Escherichia Coli,and gain the highly pure target protein of t TβRII-G and t TβRII-6 by nickel column chromatography.Observe its effect on hepatic stellate cell activited by TGFβ1 with SDS-PAGE and MTT and test the impact on the factors relating FN m RNA,α-SMA m RNA and Col-4a1 m RNA by q RT-PCR.Results: Amplified the gene fragments,t TβRII-G(393 bp)and t TβRII-6(432 bp),and identified the correct sequence what was inserted into prokaryotic expression vector by PCR,double restriction enzyme cutting and DNA sequencing.The provaryotic expression vectors,p ET-21a-t TβRII-G and p ET-21a-t TβRII-6 was constructed.Fumbled the correct inducting condition including time,temperature and the concentration of inducer.The target protein was purified and analyzed by SDS-PAGE,the most significant expression of t TβRII-G(18 k Da)and t TβRII-6(20 k Da)was under 20℃,180 rpm and 20 hours.MTT shown that the most significant impact on human hepatic stellate cell,10ng/ml and TGF-β1 activated,was by t TβRII-G and t TβRII-6.q RT-PCR shown that t TβRII-G and t TβRII-6 protein in 150μg/m L depressed the expression of the relative factors including FN m RNA,α-SMA m RNA and Col-4a1 m RNA significantly.Conclusions: Constructed the provaryotic expression vectors,p ET-21a-t TβRII-G and p ET21a-t TβRII-6 and gain the high pure protein by inducing expression.t TβRII-G and t TβRII-6 can depress the proliferation of LX-2 cell activated by TGF-β1 and decrease the function of fibrosis relative factors expression. |
