The Construction Of Eukaryotic Expressing Plasmid P～(EGFP-N1)-IL-2 And The Proliferation Of CIK In Vitro

Objective:Advances in the characterization of cytokines and tumor antigens, coupled with our increasing ability to manipulate gene expression, have fostered a new era of tumor immunotherapy . Interleukin-2 (IL-2) affects a variety of components of the cellular immune system, including B cells and macrophages by inducing the secretion of tumor necrosis factors (TNF)αandβand interferon-γ. Mainly, IL-2 is responsible for the proliferation of T cells. In animal and some human studies, systemic administration of IL-2 has antitumor effects, mediated by cytotoxic effector cells (such as lymphokine-activated killer– LAK cells and cytotoxic T lymphocytes) . Such systemic administration often induces high toxicity and is shown to be inferior to local continuous production of cytokine, for recruitment of T cells. Cytokine-induced killer cells (CIK) are non-major histocompatibility complex-restricted cytotoxic lymphocytes generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody, interleukin (IL)-2, IL-1 and interferon gamma (IFN-γ). CIK represent cells with high antitumor cytotoxicity in vitro and in vivo. CIK cells possess enhanced cytotoxic activity as compared to standard lymphokine activated killer (LAK) cells. CIK cells, express CD4 (45.4+/-3.2) % and CD8(47.7+/-11.0%) markers. It has been shown that NKT cells co-expressing CD3 and CD56 markers on their surface represent the major cytotoxic subset of CIK cells. These NKT cells are derived from T cells. Because of the increase in cytotoxicity and high proliferative response, CIK cells have a 73-fold increase in total lytic units per culture as compared to IL-2-stimulated LAK cells. Gene transfers of cytokine genes to CIK and tumor cells have been extensively studied. CIK cells transfected with cytokine genes have shown to induce antitumor effects.Methods:1.using primer premier 5.0 , four primers were designed and synthesized. Primer 3 was 5'terminal sense sequence of mIL-2 with a BamHⅠsite,primer 4 was 5'terminal antisense sequence of mIL-2 with a KpnⅠsite.2.Total RNA isolation: Total RNA was extracted from the spleen of C57 BL/6 mouse using the TRIzol Reagent.The integrity of RNA were analyzed by 1% agarose gel eletrophoresis,the ratio of 28s to 18s eukargotic ribosomal RNA should be approximately 2:1,indicating that no grass degradation of RNA has occurred.The cDNA was synthesized using total RNA primed with oligo(dT) by the SuperscriptTMⅢFirst-strand synthesis system supermix. Then IL-2 cDNA was synthesized using the synthesized first-strand primed with IL-2 specific upstream and downstream, primers by Pfu polymerase enzyme. A proper amount of the amplication products was got for 1% agarose gel eletrophoresis identify whether the amplified genes existed or not using the PCR Marker as molecular weight. 3. IL-2 DNA was cloned into PEGFP-N1 vectors and was verified by DNA sequencing.PCR products and PEGFP-N1 plasmid were digested with BamHⅠand KpnⅠ.With the role of the T4DNA ligase,connected and transformed into competent E.coli DH5a.Select transformants on LB plates containing 50ug/ml Kanamycine and extract plasmid. Whether the fusion plasmid PEGFP-N1-IL-2 construction was successful or not was proved by PCR primed with IL-2 specific primers. The PCR products was identified by 1% agarose gel eletrophoresis.The clone was sent for sequesing.4.Mix lipofectamineTM 2000 and diluted IL-2 DNA gently, then put the complexes transfect into HepG2 liver cells, after 48 hrs of incubation, then extract total RNA, using RT-PCR, the PCR products was identified by 1% agarose gel eletrophoresis to identify the expression of IL-2 DNA in HepG2 liver cells. 5.Generation of CIK. CIK cells were generated as described previously. In brief, non-adherent Ficoll separated mouse spleen mononuclear cells derived from healthy C57BL/6 mouse were prepared and grown in RPMI 1640 medium, containing 10% fetal calf serum, 25 mM Hepes, 100 U/ml penicillin and 100μg/ml streptomycin. One thousand IU/ml mouse recombinant interferonγwas added on day 0. After 24 hrs of incubation, 50 ng/ml of mouse anti-CD3 , 100 U/ml mouse interleukin-1αand 300 U/ml mouse interleukin-2 were added. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and sub-cultured every third day in fresh complete medium with 300 U/ml IL-2 at 3×106 cells/ml. CIK cells were harvested on day +7 .The phenotype of CIK cells were tested by Flow cytomety using CD3-4-8.NK1.1 on day 0.day4 and day 7.Results:1.Total RNA extraction of C57BL/6 mouse spleen ,the OD260/OD280 was 1.845,the Total RNA was not antaminated by the protein and could be used cDNA synthesis. After the 1% agarose gel eletrophoresis, the Total RNA showed the 28s.18s and 5s RNA belts clearly and the 28s belt was twice as wide as 18s belt which showed that the total RNA was intact. 2.After 1% agarose gel eletrophoresis, the amplification product of the extracted total RNA by RT-PCR can be clearly seen as a specifically amplified strip about 510bp,consistent with the expected segment in size.3.After the construction of the infusion plasmid PEGFP-N1-IL-2, IL-2 PCR primers was added into the extracted infusion plasmid template.The PCR amplification product showed clearly a specific strip about 510bp which showed that the infusion plasmid construction was successful.The cloned DNA segment in constructed PEGFP-N1-IL-2 plasmid was sequenced. It was found that the cloned C57BL/6 mouse IL-2 DNA sequence was consistent with the report in GeneBank. 4.Mix lipofectamineTM 2000 and diluted IL-2 DNA gently, then put the complexes transfect into HepG2 liver cells, after 48 hrs of incubation, then extract total RNA, using RT-PCR, the PCR products was identified by 1% agarose gel eletrophoresis showed clearly a specific strip about 510bp which identify the expression of IL-2 DNA in HepG2 liver cells. 5.The CIK cells were phenotyped with antibodies against CD3.CD8 and NK1.1 were consistent with the expected results.The percentage of CD3>90%,CD8>30%,NK1.1>10%.Conclusion:1.Cloning and sequencing of C57BL/6 mouse IL-2 DNA provided the material foundation for study mIL-2. 2.The infusion plasmid PEGFP-N1-IL-2 was successfully constructed and provide premise for studying the eukaryotic expression of the mIL-2. 3. the IL-2 DNA was expressed in HepG2 liver cells. 4.The CIK cells were phenotyped with antibodies against CD3.CD8 and NK1.1 were consistent with the expected results.The percentage of CD3>90%,CD8>30%,NK1.1>10%.