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Expression Studies of Anti-CD28 ScFv in Sf9 Cells and Silkworm, Bombyx Mori

Author: HuangGuangMei
Tutor: ZhuJiang
School: Suzhou University
Course: Biochemistry and Molecular Biology
Keywords: CD28 ScFv Bac-to-Bac expression system solubilization silkworm, Bombyx mori
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 55
Quote: 1
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Abstract


The mouse anti-human CD28 ScFv gene was subcloned into Bac-to-Bac transfer plasmid pFastBacHTa, and the recombinant plasmid pFastBacHTa-ScFv was transformed into DH10Bac competent cells. The rBacmid-ScFv virus was obtained by screening of white plaque and identification by PCR. The rBacmid-ScFv DNA was transfected into Sf9 cells using lipofectin and the virus rBV-ScFv was harvested. After Sf9 cells were infected with the rBV-ScFv, the expression of protein was analyzed by SDS-PAGE and Western blot. The highly expressed specific protein was approximately 36 kDa and reached to 25.7% of total protein, resulting in the form of insoluble aggregate. The purified protein of CD28 ScFv was obtained by Ni-NTA Resin after solubilization. Flow cytometry assays showed the specific binding ability of CD28 ScFv to T-28 cells (T cell transfected with CD28 gene), which could complete with the antibody 2F5 produced by a mouse anti-Human CD28 2F5 hybridoma.HyNPV is a new type of baculovirus which was derived from recombining of BmNPV and AcNPV. In this study, silkworm-HyNPV expression system was used to express the CD28 ScFv gene.Based on design of the fusing gene of CD28 single-chain antibody, two Bac-to-Bac BEVS were applied for construction of anti-Human CD28-ScFv transfer vectors and recombinant viruses. Finally, the recombinant viruses were obtained and were expressed in SF9 cells and silkworm. In the first time, we obtained highly purified products of anti-Human CD28 single-chain antibody, and discussed insoluble problem of the products. This is the first report for high level expression of heterologous protein in aggregate form in insect cells and its subsequent solubilization and purification. This work will be helpful for the development of anti-tumor drugs.

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