Construction, Expression And Purification Of An Immunotoxin A Human Anti-c-Met Single-chain Antibody Fused To PE38KDEL And Its Killing Effect On HCC Cell Line

Hepatocellular carcinoma(HCC) is one of the most common cancers worldwideand remains one of leading causes of death from cancer in China. Despite recentadvances using convertional approaches(surgery, chemotherapy, and radiotherapy),most of human cancers remains incurable and prognosis remains poor. It is clear thatnew therapeutic approaches are urgently needed for those patients who haveunresectable cancer at the time of diagnosis.This study describes the construction and expression of a new recombinantimmunotoxin expression vector pBAD/GⅢA/c-Met/PE38KDEL ,composed of antic-Met single-chain Fv fragmen and PE38KDEL gene, then examines the cytotoxicityof the purified product(c-Met/PE38KDEL) on human HCC cell line SMMC-7721. Inorder to get a new targeted agent which is high affinities and activities to HCC butlow cytotoxities to normal cells.Methods1. Based on the sequence of c-Met Fab fragment, two pairs of primers weredesigned and synthesized for VH and VL fragment. The plasmid of pComb3XTT/c-Met Fab(AM2-26) was used as templates for polymerase chain reaction(PCR). Thenthe purified product of PCR were cloned into pMD-18T vector, and the recombinants were sequenced.2. Having been amplified and sequenced, c-Met scFv gene was inserted intocorresponding sites of expression vector pBAD/GⅢA/ PE38KDEL, in order toconstruct the fusion gene.3. The recombinant clones were tested by the methods of restrictional enzymecut and PCR, and then were transfected into E.coli TOP10. The fusion gene wasinduced to express by L-arabinose. 12%SDS-PAGE and western blot were used toevaluate whether the protein was expressed and where the protein was expressed, inthe supernant or in the insoluble inclusion bodies. (His)6 was used as a fusion partnerto provide a"tag"for the subsequent purification. FACS test was used to evaluatewhether c-Met/PE38KDEL had the affinity with SMMC-7721.4. The apoptosis of SMMC-7721 was tested by FACS, when differentconcentration recombinant immunotoxin c-Met/PE38KDEL was added; Thecytotoxicity of the protein on SMMC-7721 was evaluated by MTT assay, and IC50was concluded.ResultResults1. Two bands of 350bp were displayed by agarose gel electrophoresis with PCRproduct and the scFv was about 750bp. The sequences of the gene were accordantwith respected.2. The recombinant clones were picked out by the methods of PCR test andrestrictional enzyme cut, and the expected bands were identified. Then the expressionplasmid of pBAD/GⅢA/c-Met/PE38KDEL were transfected into E.coli TOP10.3. The immunotoxin was induced to express by L--arabinose. 12%SDS-PAGEand western blot showed that a new protein of 70ku was expressed in E.coli whichwas in agreement with the expected molecular mass of the fusion protein of c-Met and PE38KDEL. The recombinant products were mainly insoluble inclusion bodies. Afterpurification, a single protein band of approximately 70ku was demonstrated on SDSPAGEfor the identification of the purified fusion protein. FACS indicated that c-Met/PE38KDEL had the affinity with SMMC-7721.4. FACS indicated that recombinant immunotoxin c-Met/PE38KDEL caninduce human HCC cell line SMMC-7721 to apoptosis. MTT indicated that theproliferation of SMMC-7721 which were treated with different concentrations of c-Met/PE38KDEL for 72h was remarkably inhibited. A concentration-dependentdepression activity was confirmed among the concentration of 0.001ug/ml~100ug/ml(Person goodness of fit test,χ2＝8.766,P>0.05). The relationship of c-Met/PE38KDEL concentration and SMMC-7721 inhibitory rate determined by Probitregression analysis was expressed in the equation Probit (P)=-0.27577+0.40995lg(X),and IC50 was 8.32 ug/ml.ConclusioConclusion1. The c-Met scFv gene was amplified and cloned successfully.2. The fusion gene of c-Met and PE38KDEL was constructed and induced toexpress fusion protein in E.coli.3. Purified c-Met/PE38KDEL significantly inhibited survival rate of in vitrocultured cell line SMMC-7721.