Activity Of Sle And Pbmc Of Patients With Iddm Of Th1/th2 Cytokine Gene Cloning And Sequence Analysis

Department of Molecular VirologyMedical College of Qingdao UniversityPostgraduate student Wang JingTutor Prof. Wang Bin Objective To search novel Th1/Th2 cytokines encoding genes related with systemic lupus erythematosus(SLE)and insulin dependent diabetes mellitus(IDDM)diseases. Methods 3 whole blood samples were collected from one healthy volunteer and two patients with active-phase SLE and IDDM respectively. The peripheral blood mononuclear cells (PBMC) were isolated from these samples and the total RNA were extracted from PBMC. Then 3'end anchored primers and 5' end arbitrary primers were used for differential display reverse transcription-polymerase chain reaction (DDRT-PCR) to amplify the Th1/Th2 cytokines-related gene fragments from the extracted samples.The DDRT-PCR products were displayed by silver staining method after urea-denaturing 6% polyacrylamide gel electrophoresis. Seven differential cDNA bands were displayed, named WJ1~WJ7 respectively. The differential displayed WJ2 and WJ4 bands were selected to recover from the polyacrylamide gel and reamplified by PCR. The purified reamplified products were ligased to pGEM-T Easy vector, and then transformed into the E.coli DH5αcompetent cells.The recombinant plasmids were confirmed by endonucleotidase analysis after white-blue clone selection. The plasmids from the positive clone were sequenced. The gained sequences were submitted to GenBank,EMBL and DDBJ DNA databases through Internet for homology analysis of nucleotide sequences with known sequences. Results The WJ2 fragment from IDDM patient is 380bp long, and its nucleotide sequence had 56.7% homology with IL-2; The WJ4 fragment from SLE patient is 122bp long, and its nucleotide sequence had 59.3% homology with IL-4. The two fragments weren't expressed in healthy volunteer; WJ2 fragment was expressed very weakly in SLE patient; WJ4 fragment wasn't expressed in IDDM patient. Conclusion WJ2 and WJ4 fragments may be two EST(expressed sequence tag)sequences of possible representing novel genes. The analysis of differential displayed sequences may provide valuable information for discovering novel Th1/Th2 cytokines-related genes.