Abstract:

Abstract:Objective
To obtain recombinant enzyme donor (ED) and enzyme acceptor (EA) fragments of  β-galactosidase in E.coli and establish new cloned enzyme donor immunoassays (CEDIA) for clinical use. Methods The encoding sequences of ED and EA fragments were amplified with pSV-β- galactosidase control vector as template and inserted into pGEM-Teasy vector,the target gene was chosen by double digestion,PCR and sequencing,then ED and EA fragments were inserted into the expression vector pET20b+. The competent cells of host strain of BL21 were transformed by the recombinant plasmid.The expression of the target protein was induced with IPTG and purified by Ni2+-NTA agarose column.The β-galactosidae with activity is formed.
Results The cloned fragments of ED and EA were 100% consistent with that of pSV-β-galactosidase control vector.The expression vector pET20b-ED and pET20b-EA were constructed and expressed.The target protein was purified by Ni2+ -NTA agarose column.The expressed fusion-protein ED fragment was 14 000 and EA fragment was 116 000 in SDS-PAGE as expected.Conclusion ED and EA proteins are prepared successfully and β-galactosidase with activity is formed.

Key words: β-galactosidase；enzyme acceptor；enzyme donor；immunoassay