| PurposeThrough gene engineering technique, to construct the phage display library of single-chain antibody obtained from mouse immunized with recombinant human tumor necrosis factor -a (rhTNF-a), which will dramatically decrease the immunogenic of monoclonal antibody originated from mouse. To resolve the problem of the source and the price of antibody and obtain more stable and high-quality antibody for treatment purpose. To study the receptor binding site of human rhTNF-a and the relationship between its structure and its function at advantage of the neutralization characteristic of monoclonal antibody. To explore a new method to use genetic engineering to get more the high-purity and high-vigor product, which will provide a novel kind of economic and effective antibody for diagnosis and treatment concerning TNF-α. MethodBy the technique of phage display instead of the hybridoma, the mouse were immunized with rhTNF-α, then the lymphocytes were obtained from its spleen. After the cDNA was reverse-transcribed from total RNA extracted from immunized-mouse lymphocytes, the Ig VH gene of mouse was amplified through Mouse ScFv Moudule/Recombinant Phage Antibody System(Amersham USA), with Light primer mix as the primer which is a mixture of 1 0 kinds of primers and designed to amplify the Ig VL. The heavy-chain and kappa light-chain variable region genes(VH and VL)amplified by PCR were assembled into single-chain antibody(ScFv) with overlap extension reaction by Linker-primer mix (a mixture of primer of 3 '-end of VH and 5 '-end of VL whose 24 bases at each side are complemental with 3 '-end of VH and 5 '-end of VL). When the ScFv was reamplified by PCR with RS primer mix( a mixture of 5 '-end primer of VH containing Sfi I site and 3 '-end primer of VL containing Not I site), the Sfi I site and Not I site were added to it, and the total anti-rhTNF- a ScFv gene was obtained. After being digested by Sfi I and Not I ,the ScFv was cloned into pCANTAB-5E phagemid by T4 ligase, and introduced into E.coli TGI The phagemid-containing bacterial colonies were infected with M13KO7 ,then the total surface display library of anti-rhTNF-a were established. Result1. The rhTNF-a (purchased from beijing general naval hospital) exhibited no other protein zone after SDS-PAGE and stained with Coomassie brilliant blue (CBB). The BALB/C mouse( female ,6 weeks old, more than 16g in weight and purchased from Military Medicine Academy of Science) were immunized with the rhTNF-a adding Forsten's adjuvant, according to the basic immune method. By ELISA, the titer of antibody in serum was up to 10-5-10-6, which meet the demand to construct the antibody library.2. The heavy-chain and kappa light-chain variable region genes amplified by RT-PCR with respective primer. After 1.5% agarose gel electrophoresis, the DNA strap of VH and VL can be distinguished easily which were around 340bp and 325bp.3. The VH and VL were assembled into ScFv with Linker primer mix, and the reamplification was performed with RS primer mix, then the productwith 750bp was added with two restriction site, one is for Sfi I at 5 '-end and the other is for Not I at 3'-end. Sfi I and Not I are rare in gene of antibody protein. The DNA fragment containing restriction site was insert into the phagemid pCANTAB-5E in front of gpIIIgene. After introduced into E. coli TGI by transporation, the VH and VL gene library of antibody of rhTNF-α was constructed.4. The gene library was remedied with the use of helper phage M13K07 in the culture medium containing ampicillin and kanamycin. Because the promotor lac of recombinant pCANTAB-5E was desuppressed, the ScFv fusion protein began to be transcribed and translated. The helper phage M13KO7 can help the phage replicate and released from the cells as a complete phage, at the top of which exhibit the single-chain fusion antibody of rhTNF-α. the phage antibody library completed was about 4.6 106, the litre of plaque was 2.1 1011 PFU.L-1 after superinfection with M13KO7. ConclusionThe total RNA of l...
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