Construction Of Prokaryotic Expression Vector Of The Restructuring Of Sika Deer Thymosin Beta4and Activity Of Study

Objective:Used the genetic engineering techniques to build the original thymosin β4(Thymosin β4,Tβ4) prokaryotic expression vector of the sika, and induced its toexpress the target protein, and then carried content determination of the purifiedpurpose protein and cell activity studies, to lay the foundation for revealling thebiology meaning of high expression of deer antler Tβ4as well as the development andapplication.Methods:Used trizol to extract of deer antler total RNA; the purpose gene of Tβ4wasamplified by RT-PCR; TA cloning, gene sequencing and construct an expressionvector pET-28a-Tβ4; expression vector was transformed into E.coli BL21host, strainwith IPTG induced expression of the target protein; used metal chelatechromatography and gel filtration chromatography purification of the target protein;Bradford assay was used to carry out the protein content; MTT assay was used toresearch active experiment of the alosteoblasts and chondrocytes.Results:1Tβ4target gene was successfully amplified, its full length of135bp, encoding45amino acids, PI was5.02, the theoretical molecul ar weight5052.65.2And constructed the pET-28a-Tβ4prokaryotic expression vector; optimized thebest IPTG induction concentration and induction time of0.5mM,4h.3The target protein by affinity chromatography and gel chromatography, afterpurification, SDS-PAGE electrophoresis resμL ts showed a single band, and themolecular size is consistent with the theoretical value; per mg protein yield was, theprotein purity was91.6%.4Via the cells activity researching, the recombinant thymosin β4had a proliferationrole both in osteoblasts and chondrocytes. Conclusion:Tβ4has significantly activity in promoting the proliferation of bone cells,recombinant Tβ4can lay the foundation for the development of high-tech sikaproducts, and should play a more important role in antler growth process.