Prokaryotic Expression, Purification Of Human Single-chain Antibody Against COX-2and Analysis Of Its Activity

Cyclooxygenase-2(COX-2) is the key enzyme which catalyzes the conversionof arachidonic acid (AA) into prostaglandins (PGs). A large number of studies haveshown that the induced expression of COX-2is one of the early hallmark events oftumorigenesis and an important promoting factor for the transformation of malignanttumor cells. COX-2plays an important role in tumorigenesis, tumor progression,invasion and metastasis process. It has been an important target for cancer treatment.How to lower the over-expression and activation of COX-2has become a popularissues. In consideration of the toxic side effects of the COX-2non-selectivenon-steroidal anti-inflammatory drugs (NSAIDs) and selective inhibitor, specific andefficient ways and strategies to inhibit COX-2biological function will be of greatsignificance.Because of small molecular weight and strong penetration of tissues, scFv hasunique advantages in the diagnosis and treatment of tumors, a variety of scFv-basedanticancer drugs have been used in clinical trials. In this study, the biologically activerecombinant human COX-2protein was expressed with prokaryotic expressionsystem. Then the anti-COX-2human single chain antibody gene was obtained fromphage display scFv library by using recombinant human COX-2protein as antigenThe soluble expression and purification of anti-COX-2human scFv was conducted.We characterize the binding activity and affinity of anti-COX-2human scFv and theinteraction between anti-COX-2human scFv and COX-2protein in tumor cells withELISA, Western blot, immunoprecipitation The details are as follows:1. pET28b-COX-2expression vector was successfully constructed andsoluble COX-2protein with biological activity was obtained. 2. Soluble anti-COX-2scFv with biological activity was obtained.. ELISA andwestern blot results showed that the anti-COX-2scFv had specific ability to bind torecombinant human COX-2protein.3. Competitive ELISA indicated that the binding between anti-COX-2scFv andCOX-2was specific.4. Noncompetitive ELISA showed that the affinity constant binding torecombinant human COX-2was (1.67±0.31)×108.5. Western blot and immunoprecipitation showed that anti-COX-2scFv can bindto COX-2in tumor cells specifically.The above results showed that we obtained COX-2with biological active andanti-COX-2human scFv with good binding active in vitro and in vivo. This study laidthe foundation for anti-tumor treatment targeting COX-2.