The cloning,expression and bioactivity of Fas activation domain / 中国免疫学杂志

ABSTRACT

Objective:To clone and express human Fas activation domain(FasAD)with the bioactivity.Methods:The FasAD cDNA was amplified by seminested RT PCR,and then inserted into the prokaryote vector of pTYB12 expressed intein protein to construct the recombinant plasmid of FasAD pTYB12.The FasAD peptide was expressed and purified in IMPACT TM CN system by the method of one step affinity purification.Results:Sequence analysis revealed that cloned FasAD cDNA sequence was completely same as that of Genebank record(M67454).Soluble fusion protein was successfully expressed by induction of IPTG.The FasAD peptide with a molecular weight of 5 000 was obtained by purification and was recognized by the rabbit anti human Fas polyclonal antibody in Western blot analysis.It’s activity of inhibition of apoptosis induced by rhFasL can each to 70% in primary biological detection.Conclusion:The above results indicated that FasAD peptide could be prepared by using the IMPACT TM CN system,thus laid a relatively experimental foundation for further research of relationship between structure, function and interaction with it’s ligands,and for further development of biological immune modulator. [