Construction Of PcDNA3.1(一)/hTGF β₃ And The Stable Expression System Of NIH3T3 Fibroblast With HTGF β₃ And The Study On The Proliferation Of The Stable Transfection Of NIH3T3 Fibroblasts

Objective 1.To construct eukaryotic expression vector of human transforming growth factor β₃(pcDNA3.1 ( - ) /TGF β₃). 2.To construct and identify the stable expression system of NIH3T3 fibroblast with hTGF P 3. 3.To verify hTGF β₃ expression in the stable expression system of NIH3T3 fibroblasts and investigate its proliferation.Methods A eukaryotic expression vector for hTGF P 3 (pcDNA3.1 (—) /TGF β₃) was constructed by use of recombinant DNA technique. The stable transfection of NIH3T3 fibroblasts with recombinant plasmid expressing hTGF P 3 was established by using Lipofectamine^TM2000 and G418 selection. The mRNA expression of TGF β 3 was detected by the RT-PCR method. MTT were adopted to examine the biology of the stable transfection of NIH3T3 fibroblasts.Results Digestion of the recombinant plasmid with BamHI and HindIII liberated DNA fragments with expected size .The sequence of DNA fragment is identifical to that published in GenBank. After G418 selection and RT-PCR analysis, 7 out of 10 cell lines transfected with pcDNA3.1 (—) /TGF β₃ expressed with very high level of TGF β₃,as compared with vector control transfectants that showed no expression, and compared with the other cell lines that expressed relatively low level. The stable transfection of NIH3T3 fibroblasts growth slowed down, especially on 4^th to 5^th days . Conclusion The eukaryotic expression vector of human transforming growth factor β₃ (pcDNA3.1 (-) /TGF β₃) and the stable expression system of NIH3T3 fibroblast with hTGF β₃ were constructed successfully . The transgene of TGF β₃ could inhibit the proliferation of NIH3T3 fibroblasts in vitro.