Construction Of The Retroviral Vector To Express HIL-2-EGFP Fusion Proteins

This study aims at constructing the retroviral vector to express the human interleukin-2(hIL-2) and the enhanced green fluorescent protein (EGFP) as fusion proteins. At first, a linker was designed and synthesized, which was 150bp. Then it was inserted into the multiple cloning sites(MCS) of pUC57. The new vector was named pUC-MCS. Secondly, we digested the pUC-MCS and pRevTet-On with the restriction endonucleases, recovered the target fragments respectively, and ligated them together. The recombinants were transformed into E.coli DH5a. After selecting the correct plasmid, we named it pRev-MCS. Then we digested the pRev-MCS and pEGFP-C1 respectively, obtained the recombinant plasmids pRevEGFP-C1 through ligating of appropriate fragments. Meanwhile, we constructed the intergradation plasmid pEGFP-C1-IL-2, by combining pBV220-IL-2 and pEGFP-C1 in the same method. At last, we digested the pRevEGFP-C1 and pEGFP-C1-IL-2 respectively, and obtained the recombinant retroviral vector pRevEGFP-C1-IL-2 which could express the fusion proteins of the hIL-2 and the EGFP. The digestion analysis proved that the retroviral vector, pRevIRES2-EGFP-IL-2, was constructed correctly. The retroviral plasmids were transfected into the packaging cells PT67 with Lipofectin. Resistant clones were survival under G418 selection. By the cultured of these clones, we obtained the packaging cells PT67/C1-IL-2 which could produce the...