Construction And Expression Of A Humanoral Recombinant Immunotoxin Against Hepatocellular Carcinoma And Its Killing Effect On HCC Cell Line

Hepatocellular carcinoma(HCC) is one of the most common cancers worldwide and remains one of leading causes of death from cancer in China. Despite recent advances using convertional approaches(surgery, chemotherapy, and radiotherapy), most of human cancers remains incurable and prognosis remains poor. It is clear that new therapeutic approaches are urgently needed for those patients who have unresectable cancer at the time of diagnosis.This study describes the construction and expression of a new recombinant immunotoxin expression vector pBAD/G III -A3/ Abrin-A ,composed of anti-MAGE-A1 single-chain Fv fragment(A3) which is screened from the humanoral ScFv antibody library gene and Abin-A gene, then examines the cytotoxicity of the purified product(A3/ Abrin-A) on human HCC cell line BEL-7402. In order to get a new targeted agent which is high affinities and activities to HCC but low cytotoxities to normal cells.Methods1. Based on the sequence of A3, anti-MAGE-Al single-chain Fv fragment which is screened from a humanoral ScFv antibody library, and the published nucleotide sequence of Abrin-A, two pairs of primers weredesigned and synthesized. The plasmid of pIT2-A3 and PUCm-T/Abrin-A were used as templates for polymerase chain reaction(PCR). Then the purified product of PCR were cloned into pMD-18T vector, and the recombinants were sequenced.2. Having been amplified and sequenced, A3 gene and Abrin-A gene were inserted into corresponding sites of expression vector pBAD/GIII, in order to construct the fusion gene.3. The recombinant clones were tested by the methods of restrictional enzyme cut and PCR, and then were transfected into E.coli TOP 10. The fusion gene was induced to express by L-arabinose. 12%SDS-PAGE and western blot were used to evaluate whether the protein was expressed and where the protein was expressed, in the supernant or in the insoluble inclusion bodies. (His)6 was used as a fusion partner to provide a "tag" for the subsequent purification. Dot-blot was used to evaluate whether A3 / Abrin-A had the affinity with MAGE-A14. The apoptosis of BEL-7402 was tested by FACS, when different concentration recombinant immunotoxin A3/Abrin-A was added; The cytotoxicity of the protein on BEL-7402 was evaluated by MTT assay, and IC50 was concluded.Results1. Two bands of 750bp were displayed by agarose gel electrophoresis with PCR product. The sequences of these two genes were accordant with other reports. A3 gene was 732bp and encoded 244 amino acids, and Abrin-A was 753bp and encoded 251 amino acids.2. The recombinant clones were picked out by the methods of PCR test and restrictional enzyme cut, and the expected bands were identified.Then the expression plasmid of pBAD/gIII-A3/Abrin-A were transfected into E.coli TOP 10.3. The immunotoxin was induced to express by L~arabinose. 12%SDS-PAGE and western blot showed that a new protein of 60KD was expressed in E.coli which was in agreement with the expected molecular mass of the fusion protein of A3 and Abrin-A. The recombinant products were mainly insoluble inclusion bodies. After purification, a single protein band of approximately 60KD was demonstrated on SDS-PAGE for the identification of the purified fusion protein. Dot-blot indicated that A3 / Abrin-A had the affinity with MAGE-A1.4. FACS indicated that recombinant immunotoxin A3 / Abrin-A can induce human HCC cell line BEL-7402 to apoptosis. MTT indicated that the proliferation of BEL-7402 which were treated with different concentrations of A3 / Abrin-A for 72h was remarkably inhibited. A concentration-dependent depression activity was confirmed among the concentration of 0.001ug/ml~100ug/ml (Person goodness of fit test, x 2 = 0.03,P>0.05). The relationship of A3 / Abrin-A concentration and BEL-7402 inhibitory rate determined by Probit regression analysis was expressed in the equation Probit(P)=:0.183-0.369X, and IC50 was 3.12ug/ml.Conclusion1. The A3 gene, anti-MAGE-Al single-chain Fv fragment(A3) which is sc...