Zhang Xuhui,He Weiwei,Tan Wenting,Dan Yunjie,Li Huiwen,Wang Yong,Deng Guohong.Construction of transgenic vector pBR002-HLA-DRB1*0803 and its expression and antigen-presenting function[J].J Amry Med Univ (J Third Mil Med Univ),2016,38(09):916-920.
Zhang Xuhui,He Weiwei,Tan Wenting,Dan Yunjie,Li Huiwen,Wang Yong,Deng Guohong.Construction of transgenic vector pBR002-HLA-DRB1*0803 and its expression and antigen-presenting function[J].J Amry Med Univ (J Third Mil Med Univ),2016,38(09):916-920.DOI:
Objective To construct a transgenic vector of human HLA-DRB1*0803 gene
and validate its expression and antigen-presenting function in vitro. Methods The cDNA encoding the HLA-DRB1*0803 gene was reverse transcribed and amplified by RT-PCR from the DRB1*0803 BLCL cell lines. The sequences of mouse Eα promoter and rabbit β-globin intron 1 were synthesized.The above 3 fragments were inserted into the plasmid pBR002-lacz
in a particular order. Successfully constructed pBR002-HLA-DRB1*0803 transgenic vector was used to transfect DCS cells and B cell line by Lipofectamine 2000
and the target proteins were detected by Western blotting and immunofluorescence assay. T cell proliferation assay were used to validate whether the transgenic DCS cells can present HBcAg antigens to human CD4
+
T cells and polarize the CD4
+
T cells. Results A pBR002-HLA-DRB1*0803 transgenic vector was successfully constructed
and verified by dual restriction enzyme and sanger sequencing. After transfection
the DCS cells and B cells expressed the target protein at a molecular weight of 29×10
3
. Immunofluorescence assay results showed that the HLA-DRB1 proteins were expressed mainly in the cytoplasm of DCS cells after transfection. T cell proliferation assay showed that the transfected DCS cells presented the HBcAg to the human T cells
resulting in a specific CD4
+
T cell response. Conclusion HLA-DRB1*0803 transgenic vector is successfully constructed
which can express the HLA-DRB1*0803 at the cellular level
present viral antigen and stimulate a HBcAg-specific CD4