Construction Of Recombinant Immunotoxin-rE/CUS And Investigation Of Its Anti-tumor Activity In Vitro

Object Targeting EGFR as a carrier,the cucurmosin as a "bullet" use genetic engineering methods to design,construct,and express a new EGFR specific recombinant immunotoxin rE/CUS and evaluate its antitumor activity in vitro.Methods1.Construction,expression,and purification of rE/CUS fusion protein based on the sequence of CUS and 7D12,this is already known by our group.A pair of primers was designed,and the appropriate restriction sites and protective bases were introduced at both ends of the primers.Polymerase chain reaction(PCR)was carried out with p ET-32a-CUS and p ET-32a-7D12 as templates respectively.After purification,the positive clones were selected.The 7D12 gene was then connected to CUS with the linker(G4S)3 by overlapping PCR.2.After agarose gel electrophoresis was performed,the amplified products were digested,purified,ligated between Ndel and Xhol site of plasmid vector p ET-32 a,and linked with the target fragment and vector.The fusion gene rE/CUS was cloned into E.coli expression vector,p ET-32a(+).A prokaryotic expression system was used to express the protein,the solube protein was purified using His-tagged fusion protein kit according to IMAC.3.SDS-PAGE and Western blot was used to identify the protein.4.EGFR expression on cell lines(HepG2,A549,SW116 and SW620)and rE/CUS binding capability was detected by using FACS Calibur(BD Biosciences).5.The antitumor activity of recombinant immunotoxin rE/CUS was detected by SRB.6.Flow cytometric was used to analyze tumor cell lines Hep G2,A549 apoptosis induced by different concentration of rE/CUS.7.Western blot was used to detect the protein expression level of PI3K?AKT?m TOR?P70S6K?Bax?Bcl-2?Caspase 9?Caspase 3 in Hep G2 cell after rE/CUS intervence.Results1.PCR amplification products which were detected by agarose gel electrophoresis show a 1100 bp specific expected band,sequencing results showed that the rE/CUS full-length gene 1161 bp,encoding 386 amino acids,indicating that the recombinant plasmid was constructed successfully.2.Recombinant immunotoxin expression induced by IPTG and obtained with polyhistidine fusion protein(his)6,sonication showed the protein expressed with inclusion bodies and soluble protein,Ni affinity chromatography was used to purified the protein and 5mg obtained soluble protein per liter of broth.3.rE/CUS bind with EGFR highly expression tumor cell lines human hepatocellular carcinoma cell(Hep G2),non-small cell lung cancer(A549),and colon cancer cells(sw116),but not binding to EGFR negative cells SW620.4.The results of SRB showed that 7D12 alone did not significantly inhibit the proliferation of EGFR highly expression tumor cell lines(Hep G2,A549 and SW116)and EGFR negative cell line SW620.Recombinant CUS(r CUS)had a certain inhibitory effect on the four tumor cell lines,72 h effect of IC50 was(466.3±0.483)nmol/L,(716±0.760)nmol/L,(208.33±0.056)nmol/L and(263±0.044)nmol/L respectively.The inhibitory effect of rE/CUS on the proliferation of Hep G2,A549 and SW116 was obviously,which was dose-dependent and time-dependent,72 h effect of IC50 was(2.15±0.226)nmol/L,(18.33±0.018)nmol/L and(2.33±0.032)nmol/L respectively.But SW620 cell proliferation inhibition is weak,the 72 h effect of IC50was(463±0.086)nmol/L.5.Flow cytometry showed that rE/CUS could induce the apoptosis of Hep G2 and A549.On Hep G2,the apoptosis rate of rE/CUS induced by 32 nmol/L was 48%,and On A549 the apoptosis rate of rE/CUS induced by 10 nmol/L was about 56.3%.6.Western blot showed that different concentrations of rE/CUS on Hep G2,PI3 K,Akt,m TOR,P70S6 K,Bcl-2 protein expression gradually weakened,Bax,Caspase 9,cleaved-caspase3 protein expression gradually increased,showing a concentration dependent.Conclusion1.In this study,we successfully constructed the expression vector of recombinant immunotoxin rE/CUS,expression and purification of rE/CUS.2.rE/CUS could significantly inhibit the proliferation and induce apoptosis of EGFR overexpressing tumor cell lines,rE/CUS is specifically cytotoxic to EGFR overexpression tumor cell lines in vitro.3.The mechanism of rE/CUS induced apoptosis in Hep G2 cells may be related to down-regulation of the expression of PI3 K,Akt,m TOR,P70S6 K,Bcl-2 protein,activation of caspase9,and ultimately activation of downstream Caspase3.4.Our results exhibited that rE/CUS is a potential therapeutic strategy in treating EGFR-positive solid tumors.