Activity Analysis And Construction Recombinant Procaryotic And Adenovirus Vector Of IL-24,Proteomics Study Of AdIL-24

Human IL-24 was initially discovered using a cDNA library differential gene expression subtraction screening strategy to identify and clone genes upregulated during melanoma cell differentiation, biochemical data demonstrating the protein has cytokine properties, it was a number of IL-10 gene superfamily. Some studies observed that IL-24 selectively suppressed the growth of a broad spectrum of tumor cells, without affecting growth of normal cells, it can also function as a potent immunostimulatory cytokine and has immune-modulating activity, therefore it become a hot spot of tumor gene therapy. In order to elucidate the effects and the mechanisms of IL-24 on human tumors, the effects of IL-24 on cell proliferation, apoptosis of human CaSki cervical cancer cells were investigated by the recombinant protein or gene carried by adenovirus, furthermore, the proteomics approach was applied to study the effects of AdIL-24 on the CaSki cervical carcinoma cells. Part one Construction Recombinant prokaryotic vector of expressing Interleukin-24 and Analysis of its biological activity in vitro ABSTRACT Objective: To clone human IL-24 gene and construct its prokaryotic expression vector, express purify the fusion protein GST-IL-24, then study its activity. Methods: IL-24 mRNA was expressed in HeLa cervical cancer cells which were induced by mezerein. IL-24 cDNA was amplified by RT-PCR and then was subcloned to the vector of pGEX-4T-2. The mutants (G→A, T→C in IL-24 cDNA) result in the change of IL-24 protein(A75T, H124Y). Correction of mutations was performed by a two-step PCR reaction. The correct recombinant vector was transformed into E.coli BL21(DE3) and induced to express fusion protein GST-IL-24 with isopropylβ-D- thiogalactopyranoside(IPTG), the GST-IL-24 fusion protein was purified by GSTrapTM FF column and identified by SDS-PAGE and Western blot, CaSki cells were divided into three groups(PBS control group, GST group, GST-IL-24 group), the effect was detected by MTT and flow cytometry assay. Results: We get human IL-24 cDNA, the sequence of IL-24 gene was identical with that in GenBank(ac.no:NM₀06850) after correction. The prokaryotic expression plasmid pGEX-IL-24 was constructed successfully. Dissolubily GST-IL-24 was effectively induced by 0.1mmol/L IPTG, molecular weight was nearly 50 kDa. Proliferation was obviously inhibited, the inhibition rate significantly increased at 72h after treated by GST-IL-24(5mg/L, 10mg/L, 25mg/L, 50mg/L) as compared with control group: 18.1%, 29.1%, 47.9%, 56.1%. The rate of early apoptosis by Annexin V-FITC/PI flow cytometric evaluation from the CaSki Cells in GST-IL-24 group at 48h after treated was significantly higher than that of other groups(p<0.01). Conclusion: The sequence of Interleukin-24 cDNA was consistent with the known sequence from the GenBank. GST-IL-24 protein was successfully purified. It caused a dose-dependent inhibition of CaSki cells proliferation and promoted apoptosis. Part two Construction Recombinant adenovirus vector of expressing Interleukin-24 and Analysis of its biological activity in vitro ABSTRACT Objective: construct human IL-24 adenovirus vector, get recombinants and to study its biological activity. Methods: pGST-IL-24 as template, the fragment of IL-24 was acquired by PCR amplification and cloned into the the vector of pAdTrack-CMV. Generation of recombinants by cotransforming the Pme I-cut shuttle plasmids with pAdEasy-1 backbone vectors in BJ5183, Virus was multiplied in HEK 293 cells and identified by PCR and Western blot. The inhibitory effect of AdIL-24 to CaSki cells was detected by MTT assay and cell survival analysis; apoptosis was detected by Hoechst 33342 staining test, flow cytometry. Results: The pAd-IL-24 plasmid was digested by Kpn I and Xho I into two fragments, about 650bp and 9kb, the results show that we have successful cloned the IL-24 gene into the pAdTrack-CMV vector. Two fragments (about 4.5kb and 30kb) were got after digested by Pacâ… , the restriction analysis confirmed that correct recombinant adinoviral plasmid was constructed. The fluorescence was observed in 293 cells after transfection. The expression of the IL-24 protein was comfirmed by Western Blot. MTT analysis showed that AdIL-24 caused a dose-dependent inhibition of CaSki cells proliferation. Cell survival analyses indicated AdIL-24 could evidently suppress cells long-time growth. Hoechest 33342 staining test showed the apoptotic cells were characterized by cellular shrinkage, membrane blebbing, and nuclear condensation. After 48h of treatment by AdIL-24(100pfu/cell), flow cytometric results were 33.58±3.31(AdIL-24 group), 2.63±0.63(Advec group), 2.32±1.42(PBS group), group treated by AdIL-24 was significantly difference than other groups(p<0.01). Conclusion: The adenovirus vector of hIL-24 was successfully constructed and adenovirus recombinants had biological activity. Part three Proteomics Analysis of CaSki Cervical Cancer Cells Treated with Adenovirus-Mediated IL-24 ABSTRACT Objective: In the present study, we have applied the proteomics approach to study the effects of Ad.IL-24 on the CaSki cervical carcinoma cells. Methods: Total proteins of AdIL-24(100 pfu/cell/48h) treated cells and untreated cells were extracted and separated by two dimensional gel electrophoresis (2-DE). Conditions (gel strip: pH3-10NL/pH4-7; SDS-PAGE concentration: 10%/12%; electrophoresis time: 6h/7h) were adjusted, the differential expression proteins were analyzed with PDQuest software, and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDl-TOF-MS) and Mascot/MS-fit database searching. The mRNA alterations of p53, BAX, AK1, GADD45γ, BCCIP, eIF-5A were confirmed by semi-quantitative RT-PCR, p53, BAX proteins were confirmed by Western blot. Results: 2-DE patterns of CaSki cells with high-resolution and reproducibility were obtained. A total of 43 differentially expressed proteins were visualized by 2-DE and silver stain. Of these, 29 proteins were identified via MALDI-TOF-MS analysis, 15 were upregulated (eg, Tumor suppressor p53, Apoptosis regulator BAX, Adenylate kinase isoenzyme 1(AK1), Growth arrest and DNA-damage-inducible protein GADD45 gamma (GADD45γ), BRCA2 and CDKN1A -interacting protein (BCCIP)) and 14 were downregulated(eg, Eukaryotic translation initiation factor 5A(eIF-5A), Protein DJ-1, Annexin V). At the mRNA level, p53, BAX, AK1, GADD45γ, BCCIP were upregulated, eIF-5A was downregulated; p53, BAX proteins were upregulated after treated by AdIL-24. Conclusion: Several proteins expressed differentially in CaSki cells after treated by AdIL-24, which take part in some signal pathways of cell proliferation, differentiation, apoptosis, etc, the antitumor effect of AdIL-24 may relate to the interplay of the above proteins.