Cloning
Expression in E.coli and Biological Activity of Human Thymosin β4
CHE Yan-Ke1, YANG Hui, LU Fan,
PU Qin, LI Ren-De1, ZHAO Zhong-Liang*
( Department of Biochemistry and Molecular Biology, Fourth Military
Medical University, Xi’an 710032, China; 1College of
Life Sciences, Lanzhou University, Lanzhou 730000, China )
Abstract The cDNA
thymosin β4 was synthesized by combining of chemical and enzymatic
methods. First, two complement fragments of thymosin β4 cDNA were
synthesized by DNA synthesizer, and then denatured, annealed and extended by
DNA polymerase. This fragment of thymosin β4 was then inserted into
the EcoRV and HindIII restriction endonuclease site of an
expression plasmid pLDH4 (a kind of E.coli plasmid) by blunt and
cohesive ligations. Finally, the recombinant plasmid which expressed thymosin β4
was screened by digestion and DNA sequencing. This recombinant plasmid highly
expressed the thymosin β4, which accounted for 30% of total bacteria
proteins. By salting out and chromatography, a 95% purity of recombinant
thymosin β4 was obtained. Biological assay indicated that the
recombinant thymosin β4 could induce lymphocyte proliferation and
differentiation.
Key words thymosin β4; gene expression;
E-rose; MTT
*Corresponding author： Tel, 86-29-3374537; e-mail, [email protected]