Preparing EA And ED Of β-galactosidase(E.coIi) By Fusion Protein And Its Function Research

Since the development of enzyme immunoassays (EIAs) in 1971,El As have found a broad area of application in detecting diagnostic biomarkers. The quality of an enzyme immunoassays depends very much on the purity of the antigen or hapten used for conjugation, the specificity of the antibody and the choice of a suitable enzyme label. Sensitive assays require a highly purified enzyme with a high turnover number, and a low detection limit for the reaction product. Diagnostic enzymes produced by recombinant DNA technology, which increased yield and quality of enzyme and simplified preparing process, improved the development of EIA greatly.P -galactosidase of E.coli which is scarce in human blood plasma and has a variety of substrates, is one of the most frequently used enzyme labels. It is used both in heterogeneous and homogeneous enzyme immunoassay. A novel homogeneous immunoassay system, cloned enzyme donor immunoassay system(CEDIA),has been developed by Henderson in 1986 on the basis of a -complementation reaction of 3 -galactosidase (E.coli) . In the CEDIA test, genetic engineering was used for the generation and selection of enzyme acceptor (EA) and enzyme donor (ED) of P -galactosidase. The a -complementation reaction of & -galactosidase EA and ED was also used in DNA cloning, protein protein interactions monitoring and expressing immunoassays studying .objectiveTo prepare EA and ED protein of P -galactosidase with GST fusion protein by pGEX-4T-2 expression system ,and study its a complementation activity. To lay a good foundation for further study of CEDIA and utilization in expression immunoassay.MethodsAn artificial synthesized DNA segment coding for residues 6-56(modified) of P -galactosidase (ED protein) was ligated to pGEX-4T-2 vector. Another DNA segment synthesized by several steps of restrict enzyme digest and ligation , coding for EA protein, was ligated to pGEX-4T-2 vector too. These two recombined plasmid were transferred into E.coli DH5 a , GST-EA and GST-ED fusion protein expressed by induction of IPTG The fusion protein was purified by Glutathione sepharose 4B affinity chromatography in one step and digested by thrombin. Ability to a -complement of the EA/ED protein was determined by the addition of ONPG Western Blot test with Rabbit to E.coli P -galactosidase PcAb as first antibody was used to verify the fusion proteins.Results1.Restrict enzyme analysis and DNA sequencing showed that objective DNA segments were correct in sequence and insertion direction.2.Both expressed fusion proteins were mainly in soluble supernatant. The yield of EA fusion protein is 1.6 mg per liter bacteria culture while ED fusion is 2.9 mg.3.Western-Blot result indicated that fusion proteins were components of P -galactosidase.4. The result of ability to a -complement testified the protein pair we prepared was highly active for a -complementation.Conclusion1. We succeeded in constructing the fusion protein plasmids of EA and ED of P -galactosidase.2. The protein obtained by this way was highly active a -complementation.