Cloning,Expression Of Thymosin Alpha 1 Gene And Its Effect On Lymphocyte Proliferation

Objectives: To express Tandem repeat 3 of thymosin alphal(Ta 1(3)) and recombinant Ref-Ta1 (Ref is a kind of P1 phage protein) with gene engineering method and to purify and identify the biological activities, the protein-encoding genes wich had been synthesized artificially were cloned into prokaryotic expression vectors to produce the Ta1(3) and Ref-Talfor high-level expression .Methods: The Ta1(3) gene and Ref-Ta1 gene using the preferred codons of E. coli were synthesized and inserted into expression plasmids pET-22b(+) and pBV220,and transformed to E.coli BL21(DE3) and DH5a,and protein expression was induced by IPTG and controlling the culture temperature respectively. The expressed interested protein could be purified by DEAE-Sepharose Fast Flow anion exchange chromatography and CM-Sepharose Fast Flow cation exchange chromatography and identified by Western-blot, and detected for biological activity by lymphocyte proliferation test. Results:The sizes of PCR-created products were approximately 280bp and 360bp respectively. After cloned into pET-22b(+) and pBV220, the inserted DNA fragments were confirmed by restricted enzyme digestion and sequencing. The positive clones transformed by recombinant plasmids were induced by appropriate inducers, and the interested protein was expressed in BL21(DE3) and DH5a,the molecular weights of which were about 10KD and 15KD respectively. The newly synthesized Ta1(3) and Ref- Tal were deposited in bacterial cell as soluble protein .The culture was harvested and the bacterial cells were lysated to release the intracellular protein,followed by purification with ion exchange chromatography. Lymphocyte proliferation test in vitro indicated that the expressed protein showed high biological activities. Conclusions: We cloned, expressed andpurified Tα1(3) and Ref-Tα1 protein successfully, which suggested that the method of expressing Tandem repeat gene and recombinant gene was practical to express short peptides. The successful expression of target protein has established a foundation for mass production and expression of other short peptides.