[Construction of anti-CD28 single chain antibody genes and expression of the ScFv in BmN cells and the larvae of Bombyx mori].
The V(H) and V(L) gene fragments of anti-CD28 mAb were combined to form anti-CD28 ScFv gene by using TP-PCR method. Sequence analysis showed that 6 x His tag was added to it for the ease of purification and the V(H), and V(L) 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. 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 purified by plaque assay and identified by PCR method. The recombinant virus, Bm-BacPAK6 CD28-ScFv, was obtained successfully. The BmN cells and the larvae of Bombyx mori were infected by the recombinant baculovirus and harvested every 24h postinfection. SDS-PAGE and Western Blotting analysis confirmed the expression of ScFv with the molecular weight of about 28 kD. The expression in BmN cells was detected 24h post infection and it peaked at 72 h, while in the larvae of Bombyx mori, the expression was detected 48 h post infection and it peaked at 120 h.
Zhu
Yan
Y
Life Sciences School, Suzhou University, Suzhou 215123, China.
Zheng
Feng-Feng
FF
Chen
Yong-Jing
YJ
Qiu
Yu-Hua
YH
Zhu
Jiang
J
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D012831:Bombyx / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D018106:CD28 Antigens / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007814:Larva / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection