Construction,Expression,Purification & Functional Characterization Of Humanized Single Chain Variable Fragments Antibody Against Cocaine And Amphetamine Regulated Transcript

Cocaine-and-Amphetamine-regulated transtript (CART) is a drug addiction-related neuropeptide which distributed in the CNS and is involved in regulating many physiologicalprocesses, including food intake and the maintenance of body weight, reward and endocrinefunctions. CART mRNA was upregulated by acute administration of cocaine oramphetamine.Objective: Construct humanized CART single-chain variable fragments antibody ( ScFv)to minimize its immunoreactivity. Express and purify the hScFv of CART. Investigate itseffect on behavioral sensitization and conditioned place preference induced by cocaine inmice.Methods: PCR amplification was used to amplify the VH and VL fragments of hScFvand the antigen binding region of CART-mScFv. Linked the three fragments and cloned itinto the prokaryotic expression vector pET-15b. Expressed CART-hSvFvs which wereinduced by IPTG and purified proteins by the method of affinity chromatography. ELISA wasused to test the affinity. The biological activity of CART-hScFvs were evaluated bybehavioral tests named locomotion ?? behavioral sensitization and conditioned placedpreference(CPP).Results: Successfully constructed 6 pET-15b-CART-hScFV expression plasmids whichnamed AH1, AH4, AH6, AH19, AH33 and AH36 respectively. Among them AH1, AH4,AH19, AH33 and AH36 could expressed as inclusion bodies in E.coli. Purified all of the 5hScFvs with the purification above 90%. ELISA was used to test the affinity and found thatnone of them was inactivity. Choosing AH33 and AH36 with the titer of 0.17ug/ml and0.04672ug/ml to do the behavioralal tests. During the experiments, using 3 different dosagewhich AH33 was 0.04mg/kg (A|~)O0.2mg/kg (A|~)l 1mg/kg and AH36 was 0.2mg/kg (A|~)l 1mg/kg (A|~)55mg/kg.In the test of behavioral sensitization induced by cocaine found that the three concentrationsof AH33 and AH36 both could inhibit the locomotion of mice. And continuous injections ofhScFV-AH33 and hScFV-AH36 in the transferred period could inhibit the transferration ofbehavioral sensitization in mice. Moreover, the parallel result was found in another testnamed conditioned place preference (CPP) induced by cocaine in mice. The result showedthat mid dosage of AH33 and mid/high dosage of AH36 could apparently change theexpression of CPP but not prevent the reinstatement of CPP in mice. Conclusions: Prokaryotic expression system expressed functional CART hScFvs. Continuous injections of hScFV-AH33 and hScFV-AH36 in the transferred period can inhibit the transferration of behavioral sensitization in mice. Single administration (i.p.) of hScFV-AH33 and hScFV-AH36 can inhibite the express of cocaine-induced behavioral sensitization and conditioned place preference induced by cocaine in mice but not effectively inhibit the reinstatement of CPP. All the above suggested that CART-hScFV-AH33/AH36 can potentially treat drug addiction.