The Experimental Study On Excretion Interleukin In Hepatic Stem Cell By Retroviral-Mediated Transfecting Of Human Interleukin-2 Gene

PART ONEClone of human interleukin-2 cDNA and construction and expressionof interleukin-2 prokaryotic expression plasmidObjective: To amplify human interleukin-2 gene from human blood by RT-PCR, and construct interleukin-2 prokaryotic expression plasmid, express the interleukin-2 protein in Eschrichia coli..Methods: We divide the PBMC from human blood, amplify human interleukin-2 gene by RT-PCR and sequence. We edit and anlysis the gene and amino-acid by DNAStar software. The recombinant expression plasmid interleukin-2 was constructed by ligating interleukin-2 gene with the PET-32a vector. The recombinant plasmid was transformed into BL-21 E.coli. The expression of interleukin-2 was induced with IPTG. Result: The interleukin-2 was constructed correctly by sequencing, and anlysis the protein structure of IL-2. The recombinant PET-32a-IL-2 plasmid was constructed correctly by sequencing and the expression of interleukin-2 fusion protein in BL-21 was confirmed.Conclusion: The interleukin-2 was constructed correctly. The recombinant PET-32a-IL-2 plasmid was obtained successfully.PART TWOConstruction of retroviral vector containing human interleukin-2 geneObjective: To construct the retroviral vector containing human interleukin-2 gene and study IL-2 expression in the packaging cell system PT67.Methods: Using DNA combination, the combination plasmid pEGFP-N1 was digestion with EcoRI and NotI restriction endonuclease enzyme. And the EGFP cDNA was obtained at the same time, then subcloned it into the vector plpcx ligated by ligation mix, and acquired plpcx-EGFP recombinants. Restriction endonuclease enzyme analysis and DNA sequencing analysis identified it. Also, interleukin-2 gene which was amplified by PCR from PET-32a-IL-2, was digestion with BamHI 和 EcoRI restriction endonuclease enzyme. And the plpcx-EGFP was digestion at the same time, then subcloned interleukin-2 gene into the vector plpcx-EGFP ligated by ligation mix, and acquired plpcx-IL-2-EGFP recombinants. Restriction endonuclease enzyme analysis and DNA sequencing analysis identified it. Thirdly, the combination plasmid PET-32a-IL-2 was digestion with BglII 和 NotI restriction endonuclease enzyme. And the IL-2 cDNA was obtained at the same time, then subcloned it into the vector plpcx ligated by ligation mix, and acquired plpcx-IL-2 recombinants. Restriction endonuclease enzyme analysis and DNA sequencing analysis identified it. Subsequently, plpcx-EGFP was transfected into the PT67 packaging cells using lipofectamine 2000 and observe it' s transfecting rate. And then plpcx-IL-2-EGFP and Plpcx-IL-2 was transfected into the PT67 packaging cells using lipofectamine 2000. After puromycin(2ug/L) selection for 10-12 days, the survived PT67 cells secreting virus stably were selected and enriched to assay virus titer using NIH3T3 cells, and named it PT67/ plpcx-IL-2-EGPF and PT67/Plpcx-IL-2. Results: (1) plpcx-EGFP recombinants was digested into 780bp EGFP fragment and about 6.3kb linear vector. plpcx-IL-2-EGFP recombinants was digested into 462bp IL-2 fragment and about 6.3kb linear vector. plpcx-IL-2 recombinants was digested into 462bp IL-2 fragment and about 6. 3kb linear vector. The sequencing analysis showed the recombinants were correct; (2) PT67/Plpcx-IL-2-EGFP and PT67/Plpcx-IL-2 cells were acquired after puromycin selection. The titer of virus in its supernatant reached 10~5 CFU/ml.Conclusion: The retroviral vector containing human interleukin-2 gene was obtained successfully. The generated retroviral vector carrier PT67/Plpcx-IL-2-EGFP and PT67/Plpcx-IL-2 could express interleukin-2 effctively, and it provides a good basisfor further research on interleukin-2 expression in hepatic stem cell. PATR THREEConstruction of hepatic stem cell excreting human interleukin-2 Objective: To construct hepatic stem cell excreting human interleukin-2 stably and effectively by retroviral-mediated transfecting of human interleukin-2 gene. Methods: (1) The highest titer of virus infected hepatic stem cell WB-F344, after puromycin (2ug/L) selection for 2 weeks, the puromycin resistance clones were acquired ,and named it WB-F344/ Plpcx-IL-2-EGFP and WB-F344/Plpcx-IL-2, (2) The expression of interleukin-2 in WB-F344/Plpcx-IL-2 cells was detected respectively with immunocyto- chemistry, Western Blot, genomic PCR, RT-PCR and IIF. Results: (1) WB-F344/ Plpcx-IL-2-EGFP and WB-F344/Plpcx-IL-2 resistance clones growth well; (2) Immunocytochemical analysis confirmed that interleukin-2 was stored in the WB-F344/Plpcx-IL-2 cells. Western Blot analysis demonstrated the interleukin-2 expressed by the WB-F344/Plpcx-IL-2 cells; The 462bp interleukin-2 fragment was amplificated in the WB-F344/Plpcx-IL-2 cells using PCR and RT-PCR, Which demonstrated the integration and mRNA synthesis of gene IL-2, while no IL-2 fragment was amplificated by control cells. The secretive human interleukin-2 was detected in the WB-F344/Plpcx-IL-2 cells by IIF.Conclusion: By retroviral-mediated transfecting human interleukin-2 gene, the hepatic stem cell excreting interleukin-2 stably and effectively was obtained. It provides a good basis for further research on immuno-gene therapy of liver cancer.Conclusions:1. The recombinant PET-32a-IL-2 plasmid was contructed correctly by sequencing and the expression of IL-2 fusion protein in BL-21 was confirmed.2. The retroviral vector plpcx-EGPF, plpcx-IL-2-EGPF and plpcx-IL-2 was obtained successfully.3. The generated retroviral vector carrier PT67/Plpcx-IL-2-EGFP and PT67/Plpcx-IL-2 could express interleukin-2 effctively.4. By retroviral-mediated transfecting human interleukin-2 gene, the hepatic stem cell excreting interleukin-2 stably and effectively was obtained. It provides a good basis for further research on immuno-gene therapy of liver cancer.