Recombinant Protein MAP30-Induced Apoptosis Of BGC-823 Cells And The Preliminary Study Of Apoptosis Mechanism

Objective:MAP30,a type-Ⅰribosome-inactivating protein(RIP), induces apoptotic cell death in various cell lines,however,the precise apoptosis signaling pathway is unknown.Here,we cloned MAP30 gene of the Momordica charantia(MC) protein and expressed and pured the MAP30 fusion protein.And then to investigate whether MAP30 induces apoptosis in BGC-823 cells in vitro and to further assess apoptotic mechanism.Method:(1) Polymerase chain reaction(PCR) was used to amplify the MAP30 gene from bitter guard genome DNA.Then the target gene was inserted into the vector pGEM-T and identified by restriction digestion and DNA sequencing.Then the MAP30 gene fragment was inserted directionally into expression vector pET-28a.The pET-28a-MAP30 expression vector was transformed into E.coli BL21.The his- MAP30 fusion protein was expressed in E.coli BL21 with Isopropylβ-D-1-thiogalacto- pyranoside(IPTG),the target fusion protein was purified by Ni-column,and identified by SDS-PAGE.(2) Recombinant MAP30-fusion protein and human gastric cancer cells BGC-823 were co-cultured,the morphological change of cell and the effect of MAP30 on cell proliferation were observed by light microscope and the MTT test,respectively.The MAP30-induced apoptosis of BGC-823 cells was determined by flow cytometry.(3) To further investigate the mechanism of MAP30-induced apoptosis of BGC-823 cells,the mitochondrial membrane potential,the mRNA expression of caspase-9 and Apaf-1,the activity of caspase-3 and caspase-12,NF-κB expression were detected by many techniques such chemoluminescence,immunohistochemistry,RT-PCR and so on.Results:(1) The recombinant plasmid was constructed.DNA sequence analysis showed the sequence of MAP30 was 861bp.It has homology with DQ643967,DQ643968 and S79450(2) The prokaryotic expression vector pET-28a-MAP30 was successfully constructed and recombinant MAP30 fusion protein was obtained,with a relative molecular weight of 30kDa.(3) After induced by different concentrations of MAP30 fusion protein,the cells were observed obvious morphological changes;We also investigated that MAP30 inhibited the growth of BGC-823 cells in a dose and time-dependent manner,the apoptosis peak of the hypodiploid was more evident;The mitochondrial membrane potential was reduced when cells were exposed to MAP30,caspase-9 and Apaf-1 mRNA expression and the activity of caspase-3 increased,caspase-12 showed no significant change. (4) MAP30 fusion protein could activate nuclear transcription factor NF-κBConclusion:(1) The prokaryotic expression vector pET-30a-MAP30 was constructed successfully and the fusion protein MAP30 was obtained from E.coli BL21.(2) Recombinant MAP30-fusion protein inhibited the growth of BGC-823 cells in a dose and time-dependent manner.(3) The cytotoxicity of MAP30 toward human BGC-823 cells was attributable to apoptotic cell death in vitro via the mitochondrial pathway.(4) MAP30 fusion protein could activate nuclear transcription factor NF-κB.