Construction Of Transfer Vectors Containing Anti-CD28 Single Chain Antibody And Chimeric Antibody Genes And Expression Of The ScFv In BmN Cell

CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. Earlier results indicated that the monoclonal antibody against CD28 can be used for the therapies of tumour, autoimmune diseases and reduce the transplant repellency. However, long-term application decreases therapeutic effects because of the production of the human-anti- mouse antibody. Anti-CD28 mAb must be rebuilt before it can be clinically used. In this study, CD28 chimeric antibody and single chain antibody were constructed, the recombinant baculovirus transfer vectors pAcUW3/CD28- vH-γ1,vL-κand pBacPAK8/ CD28-ScFv were obtained, and the ScFv was expressed in BmN cell. The main results and conclusions are as followed:1. The genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both ends of the target genes, and inserted into the baculovirus transfer vector pAcUW3. The recombinant baculovirus transfer vector pAcUW3/CD28-vH-γ₁,vL-κwith two chimeric genes was successfully constructed.2. By using TP-PCR method, the VH and VL gene fragments of anti-CD28 mAb were combined to form anti-CD28 ScFv gene. Sequence analysis showed that 6×His tag was added to it for the ease of purification and the VH and VL gene fragments were connected by a linker containing 15 amino acids which are biased by the baculovirus promoter, ph. Then ScFv gene fragment was inserted into baculovirus transfer vector pBacPAK8. The recombinant transfer vector, pBacPAK8/CD28-ScFv was constructed successfully.3. The pBacPAK8/CD28-ScFv and the linear Bm-BacPAK6 were co-transfected into the cell line of Bombyx mori (BmN) with the help of Lipofectin, then the product was...