[Construction of a recombinant baculovirus transfer vector with two promoters expressing the anti-human CD28 chimeric antibody by using TP-PCR method].
CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.
Zhu
Yan
Y
Life Sciences School, Suzhou University, Suzhou 215006, China.
Chen
Yong-Jing
YJ
Qiu
Yu-Hua
YH
Zheng
Feng-Feng
FF
Zhu
Jiang
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D019680:Trinucleotide Repeat Expansion
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins