Construction Of Human Antisense CD40 RNA Eukaryotic Expression Vector And Its Effects On B Lymphocytes

Aim To construct human antisense CD40 RNA eukaryotic expression vector andinvestigate its effects on functions of B lymphocytes.Methods Partial sequence of Human CD40 gene was obtained by RT-PCR. Then,the PCR product was cloned with a T-A clone technique to constructCD40/pUCm-T. According to the result of sequence analysis, the PCR productwas subcloned to form CD40/pcDNA3. The recombinant was identified by dualenzyme digestion and the direction of CD40/pcDNA3 was analysed with T7primer. After being packed by lipofectamineTM2000, the recombinant wastransfected into B lymphocytes. CD40 expression on membrane, cellproliferation and antibodies concentration were detected with flowcytometry,MTT colorimety assay and EL1SA, respectively.Result 1, Human antisense CD40 RNA eukaryotic expression vector wasconstructed successfully. 2, In the presence of CD40/pcDNA3, CD40expression was significantly decreased, cell proliferation and antibodiesgeneration were significantly restrained, compared to that of thecontrols (P<0.01).Conclusion Human antisense CD40 RNA eukaryotic expression vector wasconstructed successfully and can significantly inhibit CD40 expression,cell proliferation and antibodies generation. CD40 gene plays an importantrole in both cell proliferation and antibodies generation, and antisenseRNA may be served as an effective means of regulating immune function.