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Information Journal Paper

Title

CLONING AND EXPRESSION OF CD19, A HUMAN B-CELL MARKER IN NIH-3T3 CELL LINE

Pages

  39-44

Abstract

 Background: CD19 is a pan B CELL marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study.Methods: Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic.Results: Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively).Conclusion: Stable cell surface expression of human CD19 antigen in a murine NIH- 3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera.

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    Cite

    APA: Copy

    ABBASI KENARSARI, HAJAR, SHAFAGHAT, FARZANEH, BARADARAN, BEHZAD, MOVASSAGHPOUR, ALI AKBAR, SHANEHBANDI, DARIUSH, & KAZEMI, TOHID. (2015). CLONING AND EXPRESSION OF CD19, A HUMAN B-CELL MARKER IN NIH-3T3 CELL LINE. AVICENNA JOURNAL OF MEDICAL BIOTECHNOLOGY (AJMB), 7(1), 39-44. SID. https://sid.ir/paper/313859/en

    Vancouver: Copy

    ABBASI KENARSARI HAJAR, SHAFAGHAT FARZANEH, BARADARAN BEHZAD, MOVASSAGHPOUR ALI AKBAR, SHANEHBANDI DARIUSH, KAZEMI TOHID. CLONING AND EXPRESSION OF CD19, A HUMAN B-CELL MARKER IN NIH-3T3 CELL LINE. AVICENNA JOURNAL OF MEDICAL BIOTECHNOLOGY (AJMB)[Internet]. 2015;7(1):39-44. Available from: https://sid.ir/paper/313859/en

    IEEE: Copy

    HAJAR ABBASI KENARSARI, FARZANEH SHAFAGHAT, BEHZAD BARADARAN, ALI AKBAR MOVASSAGHPOUR, DARIUSH SHANEHBANDI, and TOHID KAZEMI, “CLONING AND EXPRESSION OF CD19, A HUMAN B-CELL MARKER IN NIH-3T3 CELL LINE,” AVICENNA JOURNAL OF MEDICAL BIOTECHNOLOGY (AJMB), vol. 7, no. 1, pp. 39–44, 2015, [Online]. Available: https://sid.ir/paper/313859/en

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