J4 ›› 2010, Vol. 36 ›› Issue (1): 40-44.
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XU Shu-Fen, LIU Lei, SUN Mu-Nan, ZHAO Shuai, FANG Yan-Qiu, TAN Yan
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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 Ni2+-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 Ni2+ -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
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XU Shu-Fen, LIU Lei, SUN Mu-Nan, ZHAO Shuai, FANG Yan-Qiu, TAN Yan. Cloning| expression and activity assay of ED/EA of β- galactosidase in E.coli[J].J4, 2010, 36(1): 40-44.
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http://xuebao.jlu.edu.cn/yxb/EN/Y2010/V36/I1/40
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