Construction Of Interleukin-10-Adenovirus Recombinant Vector, Preparation Of Recombinant Virus And Analysis Of Expression Product

BACKGROUND Interleukin 10 (IL-10) is a multifunctional cytokine produced by T cells and other cell types. It regulates activities of a variety of the cells of the immune system and may play a crucial role in cross-regulation of different types of T cells. It has been reported that IL-10 has many pharmacological activities against many diseases such as self-immune, cancer and severe infections. Several inflammation cytokines are now in development for the treatment of asthma and other allergic diseases such as Interleukin-2, Interleukin-4 and Interleukin-5, but Interleukin-10 which has many special suppression functions is comparatively little considered in the treatment of asthma diseases.AIM To obtain a recombinant adenovirus which can express active mIL-10, for the further use in the treatment of asthma and other allergic diseases in gene therapy.METHODS Clone mIL-10 fragment from stimulated mouse spleen cells. Legate mIL-10 to adenovirus vector with the help of pShuttle plasmid, and detect the positive clone by PCR and restriction-enzyme digestions. Evaluate the influences of the pH value, the purity of the plasmid DNA, the cultured time before shocking and the glycerol-shock time to the calcium phosphate transfection efficiency, then transfect HEK 293T cells by optimal calcium phosphate method by using mIL-10 vector. Prepare mIL-10 recombinant adenovirus by multiple infections, then detect the virus stock by PCR and determine the virus titer by End-Point' Dilution Assay.Determine the concentration and activity of expressed mIL-10 by ELISA.RESULTS (1) Sequencing proves that mll-10 cloned from mouse spleen cells is identical to the sequence in the GeneBank. PCR using specific primers derived from adenovirus vector results a right band at 312bp, and both Xho I digestion and Pl-Sce l/l-Ceu I double digestion can also prove a successful construction of mIL-10 recombinant adenovirus vector.(2) The results show that, the best cultured time before shocking is 6 hours, which makes the transfection efficiency increase by 584. 4% and the best time of glycerol-shock is 4. 5 minutes, making the efficiency increase by 130. 8% compared to the negative group. The pH value, of 6. 96 and the pure plasmid DNA are also a key factor, affecting the efficiency of transfection significantly. The transfection efficiency to the HEK 293T cells ranges from 8.91CT3 to 2. O10"2.(3) CPE is evident after infecting cells several times, and a right band at 312bp can be observed after PCR, all of which prove a successful preparation of recombinant adenovirus stock. The virus titer is 2. 5X 107pfu/ml determining by End-Point Dilution Assay.(4) ELISA shows the recombinant virus produces active mIL-10 in 293 cells.CONCLUSION A mIL-10 recombinant virus vector ' is constructed successfully. It transfects HEK 293T cells in high efficiency by optimizing several influences to calcium phosphate transfection, and expressed mIL-10 protein with activity is detected then which reveals a triumphant preparation of recombinant adenovirus. The preparation of mIL-10 recombinant adenovirus establishes a good foundation for the further use in asthma animal models and the treatment for other allergic diseases in gene therapy.