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Development of anti-human CD271 monoclonal antibody

Author: XuYanHong
Tutor: ZhangYuGuang
School: Peking Union Medical College , China
Course: Immunology
Keywords: CD271 RT-PCR DNA immunization Monoclonal antibodies Mesenchymal stem cells
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 32
Quote: 0
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Abstract


The purpose of the experiment : the use of DNA immunization prepared anti-human CD271 molecule monoclonal antibody to investigate whether it can become used to identify the separation between the mesenchymal stem cell - specific markers . Experimental methods : RT-PCR method grams drop CD271 coding region of the full-length gene amplified genes were introduced into the eukaryotic expression vector pcDNA3.1 and prokaryotic expression vector pET22b . (1) using the method of genetic immunization , the recombinant plasmid pcDNA3.1/CD271 by intramuscular immune BALB / C mice , and each time before DNA immunization of mice quadriceps 1 week ago following pretreatment ① DNA immunization 15 minutes before the injection of venom of myocardial toxins ② DNA immunization injection of 25 % hypertonic sucrose solution . Fusion 3 days before spleen , strengthen the immune once , after using the the traditional lymphocyte hybridoma technique to prepare anti- CD271 molecule monoclonal antibody . 7 days after each immunization by flow cytometry for antibody secretion in the mouse serum . Application subclass antibody sandwich ELISA method by flow meter ( FACS ) , fluorescence microscopy to verify the binding activity of the antibody with natural membrane protein , Western blot to verify the specificity of the antibody . ( 2) the amplification of the gene into the prokaryotic expression vector pET22b construct recombinant prokaryotic expression vector pET22b/CD271 . Experimental results : (1) amplification of the the CD271 length gene 1281bp, the constructed pcDNA3.1/CD271 recombinant plasmid . (2) FACS serum titer after DNA immunization is up to 1:160 , strengthen the immune spleen titer of 1:320 . (3) by cell fusion , screening and cloning culture final two monoclonal antibody 487E and 487D . ( 4 ) two anti- Hugh subclasses are IgM, light chains are K -type . ( 5 ) Immune competition experiments show that the 487E and CD271 (BD Company ) recognize different epitopes . ( 6 ) FACS , immunofluorescence microscopy and Western blot showed that natural antibody and cell membrane proteins and degeneration of membrane proteins have good binding activity and specificity . ( 7) successfully constructed prokaryotic expression vector pET22b/CD271 . Experimental conclusion : for the the CD271 molecule monoclonal antibody 487E between the source and the bone marrow and umbilical cord mesenchymal stem cells have good binding activity , which is possible between the two sources of mesenchymal stem cells in a large number of effective separation and identification .

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