Wei He

Southern Medical University, Shengcheng, Guangdong, China

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Publications (7)3.61 Total impact

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    ABSTRACT: To construct a personalized full-length fully human antibody mammalian display library for children with systemic lupus erythematosus (SLE). The total RNA was isolated from the PBMCs of SLE children. The heavy chain variable region and kappa light chain (VH and LCκ) of the antibody genes were amplified by RT-PCR and inserted into the pDGB-HC-TM vector separately to construct the heavy chain and light chain libraries. The library DNAs were transfected into 293T cells and the expression of full-length fully human antibody on the surface of 293T cells was analyzed by flow cytometry. Using 0.8 µg total RNA as the template, the VH and LCκ were amplified and the full-length fully human antibody mammalian display library was constructed. The VH and LCκ gene libraries had a size of 9.4×10(4) and 8.4×10(4), respectively. Sequence analysis of 10 clones randomly selected from the VH and LCκ gene libraries each showed that 8 heavy chain clones and 7 light chain clones contained correct open reading frames, and flow cytometry demonstrated that all the 15 clones express full-length antibodies on 293T cell surfaces. 293T cells co-transfected with the VH and LCκ gene libraries expressed the full-length antibodies on the cell surface. The personalized full-length fully human antibody library for SLE children constructed allows display of the full-length antibodies on mammalian cell surfaces, thus providing a valuable platform for analyzing the autoantibodies, their etiological role, and their clinical implications in SLE.
    Nan fang yi ke da xue xue bao = Journal of Southern Medical University 08/2012; 32(8):1082-7.
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    ABSTRACT: To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries. Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry. The libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10). The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.
    Nan fang yi ke da xue xue bao = Journal of Southern Medical University 08/2011; 31(8):1369-73.
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    ABSTRACT: A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.
    Acta Biochimica et Biophysica Sinica 03/2011; 43(3):232-8. · 1.81 Impact Factor
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    ABSTRACT: To construct a mammalian cell surface display library of full-length human antibodies. The total RNA was isolated from human peripheral blood mononuclear cells (PBMCs), and the genes encoding the heavy chain variable regions and kappa light chains (VH and Cκ) of the antibodies were amplified by RT-PCR. The amplified VH and Cκ gene sequences were separately inserted into the vector pDGB-HC-TM. The ligation mixtures were transformed into competent E.coli DH5α cells to construct the antibody libraries, and the library sizes and diversity were analyzed. The library DNAs were transfected into CHO cells and the expression of the full-length human antibodies on the surface of CHO cells was analyzed by flow cytometry. The heavy chain gene library constructed showed a diversity of 2.6 × 10(5), and the kappa light chain gene library had a diversity of 2.0 × 10(5). Sequence analysis of 10 clones randomly selected from the constructed heavy chain gene library and 10 from the light chain gene library showed that 8 heavy chain clones and all 10 light chain clones contained correct open reading frames. Flow cytometry demonstrated that all the 18 clones expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces was detected with the positive cells reaching 31.5. A full-length human mammalian display antibody library with a combinatory diversity of 5.2 × 10(10) can be constructed in two weeks, which allows the display of full-length antibodies on mammalian cell surface.
    Nan fang yi ke da xue xue bao = Journal of Southern Medical University 02/2011; 31(2):308-12.
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    ABSTRACT: Development of a versatile mammalian display system is essential for the selection of functional human antibodies with high affinities. Here we described a novel strategy for rapid construction of full-length antibody libraries that could be efficiently expressed on mammalian cell surfaces. The universal vector pDGB-HC-TM was constructed by inserting multiple cloning site unique sequences recognized by restriction endonucleases BsmBI, SfiI, and BstXI for the pop-in and pop-out of genes of interest. Cytomegalovirus promoter, a commonly used promoter for high expression of proteins in a variety of mammalian cells, was used to drive expression of the inserted antibody genes and a transmembrane domain from platelet-derived growth factor receptor was fused in frame to the C-terminus of heavy chain consistent region to anchor the antibody expressed on the mammalian cell surface. Using this strategy, we constructed a full-length human antibody display library. DNA sequence analysis and expression analysis indicated that the library constructed had a combinatory expressible, detectable diversity of 6.58 x 10(10).
    Acta Biochimica et Biophysica Sinica 08/2010; 42(8):575-84. · 1.81 Impact Factor
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    ABSTRACT: To construct human renal cell carcinoma patient-specific full-length antibody library using mammalian cell surface display technique. Peripheral blood mononuclear cells (PBMC) were isolated from patients with renal cell carcinoma. The repertoires of kappa light chain (LCkappa) and heavy chain variable region (VH) of antibody were amplified by RT-PCR. The LCkappa and VH libraries were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO10 to construct the renal cell carcinoma patient-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length human antibodies expressed on the surface of 293T cells were analyzed by flow cytometry. The libraries of renal cell carcinoma-specific antibody kappa light chain (LCkappa) and heavy chain (IgG1) were constructed. The expression of the full-length human antibodies on the surface of 293T cell was confirmed by flow cytometry. The libraries showed an expressible combinatory diversity of 7.5x10(10). The expressible antibody library provides a useful platform for screening of renal cell carcinoma-specific antibodies.
    Nan fang yi ke da xue xue bao = Journal of Southern Medical University 05/2010; 30(5):1059-62.
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    ABSTRACT: To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells. The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed. The transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments. In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.
    Nan fang yi ke da xue xue bao = Journal of Southern Medical University 01/2010; 30(1):111-3.

Publication Stats

13 Citations
3.61 Total Impact Points

Institutions

  • 2010–2011
    • Southern Medical University
      • • School of Pharmaceutical Sciences
      • • Institute of Genetic Engineering
      Shengcheng, Guangdong, China
    • Nanfang Hospital
      Shengcheng, Guangdong, China