Construction And Purification Of A Recombinant Immunotoxin Composed Of PE38 And Humanized ScFv Against Hepatocellular Carcinoma

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 conventional 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 the those patients who have unresectable cancer at the time of diagnosis.Differentiation antigens, hormones and growth factor receptors, and viral antigens are expressed on the surface of a variety of malignant and dysfunctional cells in humans. So antibodies, recombinant hormone and growth factors, and recombinant soluble viral glycoprotein receptors have been generated that can target diseased human cells in vitro and in vivo. Gene engineering progress and itsapplication in antibody technology, makes diagnosis and targeted therapy ofmalignancy possible and has inspiring results during the last decades. Targeted therapy has become a major part of biotherapy of cancer after the conventional therapeusis.The generation of specific anti-tumor antibodies of small molecular weights, especially recombinant single-chain antibodies(scFv) make targeted therapeutic trials of cancers more attractive than ever. Highly potent peptide toxins or their subunits have been linked to ligands such as scFv and growth factors by DNA techniques to produce a new class of cancer therapeutic agents called recombinant immunotoxins. Ongoing clinical studies in patients with malignancies show high specificity and activity of these agents.This study describes the construction and expression of a new recombinant immunotoxin expression vector pGEX4T-1hscFv-PE38 ( pGEXh-PE38 ) composed of a truncated form of pseudomonas exotoxin A( PE38 )gene and a humanized rodent anti human hepatocellular carcinoma (HCC) single-chain Fv fragments(hscFv) gene, then examines the cytotoxicity of the purified product(hscFv-PE38) on human HCC cell line SMMC-7721.In order to get a new targeted agent with high affinities and activities to HCC but low cytotoxities to normal cells.Related methods and results are: The PE38 gene fragment with proper restriction enzyme sites was amplified by polymerase chain reaction(PCR),and introduced into a GST fusing protein expression plasmid inserted hscFv(humanized single-chain Fv fragments ). The recombinant vector was identified without mutation by restriction endonucleases digestion and DNA sequencing.The PE38 and hscFv is linked by nine base pares (CTTAAGAAA).After transformed into Kcole JM109 and induced by IPTG, the expected recombinant protein was expressed with molecular weight being 90kD.In different concentrations of IPTG inducement, the main product was ininclusion bodies, amounted to 9% toll% of the total host bacterial proteins. The inclusion bodies was denatured, renatured and refolded, then resolvable protein was purified by GST affinity chromatography.Desired hsdFv-PE38(66KD) with satisfied purity was obtained after cleaving fusion protein with thrombin. The cytotoxity of hsdFv-PE38 on SMMC-7721 was evaluated by MTT assay. Purified hscFv-PE38 showed obvious effect on SMMC-7721 in MTT assay , The concentrations of hsdFv-PE38 and the values of A490 showed an obvious dose-effect relationship (pearson ,x2=8. 844, v =5, p=0.115), The dose/survival rate relation was calculated as Logit P =-0.259-0.2731n(x) ,where x= concentration (ug/mL) and P =survival rate(100%) , the maximum killing rate was 75%, IC50 was 0.386g/mL.While hsdFv-PE38 did not inhibit the growth of normal liver cell line QZG in this study as comparison.The conclusion of this article are :(1)The new recombinant immunotoxin expression vector pGEXh-PE38 was constructed successfully. (2)Expression and purification of hscFv-PE38 was achieved after inducement. (3)Purified hsdFv-PE38 significantly inhibited survival rate of in vitro cultured cell line SMMC-7721. In summary ,our results demonstrate the expression strategies in this study are helpful for...