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Title

IMMUNOLOGY AND IMMUNOTHERAPY: P-75: STABLE EXPRESSION OF MODIFIED GENE ENCODING FUNCTIONAL HUMAN COAGULATION FACTOR VIII IN NIH3T3, HEPG2 AND CHO CELL LINES

Pages

  73-74

Abstract

 Objective: Hemophilia A or FACTOR VIII deficiency is a common X-linked genetic bleeding disorder in humans. Since first rFVIII there are still concerns about EXPRESSION and activity level of rFVIII in fields of industrial EXPRESSION of FVIII or gene therapy of hemophilia. FVIII contains a domainsequence organization designated A1-A2–B-A3-C1-C2 which B domain is not necessary in coagulation activity. Replacing B domain with linker sequence of both 3 and 5 end of B domain which has the RXXR sequence for furin enzyme processing, may improve secretion level of rFVIII protein.Materials and Methods: We constructed a new modified B-domain deleted FVIII cDNA and cloned into N-terminal His tagged EXPRESSION vector via GATEWAY TECHNOLOGY. B-domain containing glycosilation sites was removed in this construct but some elements were added to enhance EXPRESSION level of this recombinant rFVIII inclung Flag tag GFP linker and Furin recognition sequence. This vector transfected into three cell lines: NIH3T3 CHO and HepG2. rFVIII extracted purified and detected with SDS PAGE and western blot using anti His tag and anti FVIII antibodies and rFVIII activity was measured using ST4 kit.Results: The results showed high EXPRESSION and activity in NIH3T3 and CHO cell lines. Majority of secreted protein were proteolitically cleavaged with Furin and thus secretion level have been enhanced. Flow cytometry analyzes showed that after two weak flourscent intensity of stable rFVIII EXPRESSION NIH3T3 and CHO cells were significant. Conclusion: As a result of this Diminished glycosylation heterogeneity, B-domain-Truncated variants of FVIII which have linker containing furin processing sequence may also be a more suitable precursor for making wellcharacterized long acting FVIII variants for industrials production or gene therapy.

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    APA: Copy

    EBRAHIMI, A., SHEKARI KHANIANI, M., MANSOURI DERAKHSHAN, S., & MOHARAMI, T.. (2011). IMMUNOLOGY AND IMMUNOTHERAPY: P-75: STABLE EXPRESSION OF MODIFIED GENE ENCODING FUNCTIONAL HUMAN COAGULATION FACTOR VIII IN NIH3T3, HEPG2 AND CHO CELL LINES. CELL JOURNAL (YAKHTEH), 12(SUPPLEMENT 1 (THE 1ST INTERNATIONAL STUDENT CONGRESS ON CELL AND MOLECULAR MEDICINE)), 73-74. SID. https://sid.ir/paper/556640/en

    Vancouver: Copy

    EBRAHIMI A., SHEKARI KHANIANI M., MANSOURI DERAKHSHAN S., MOHARAMI T.. IMMUNOLOGY AND IMMUNOTHERAPY: P-75: STABLE EXPRESSION OF MODIFIED GENE ENCODING FUNCTIONAL HUMAN COAGULATION FACTOR VIII IN NIH3T3, HEPG2 AND CHO CELL LINES. CELL JOURNAL (YAKHTEH)[Internet]. 2011;12(SUPPLEMENT 1 (THE 1ST INTERNATIONAL STUDENT CONGRESS ON CELL AND MOLECULAR MEDICINE)):73-74. Available from: https://sid.ir/paper/556640/en

    IEEE: Copy

    A. EBRAHIMI, M. SHEKARI KHANIANI, S. MANSOURI DERAKHSHAN, and T. MOHARAMI, “IMMUNOLOGY AND IMMUNOTHERAPY: P-75: STABLE EXPRESSION OF MODIFIED GENE ENCODING FUNCTIONAL HUMAN COAGULATION FACTOR VIII IN NIH3T3, HEPG2 AND CHO CELL LINES,” CELL JOURNAL (YAKHTEH), vol. 12, no. SUPPLEMENT 1 (THE 1ST INTERNATIONAL STUDENT CONGRESS ON CELL AND MOLECULAR MEDICINE), pp. 73–74, 2011, [Online]. Available: https://sid.ir/paper/556640/en

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