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| Prokaryotic Expression,Purification and Preparation of Monoclonal Antibodies of Cold Inducible RNA-binding Protein in BALB/C Mice |
1.College of Animal Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, China
2.Harbin Veterinary Research Insititue, Chinese Academy of Agricultural Sciences, Harbin 15000,China |
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Abstract The CIRP gene of mouse was amplified from testis total RNA of BALB/C mouse by RT-PCR. PCR product was cloned into the pGEM-T vector to construct recombinant plasmid pGEM-CIRP for sequencing. The correct CIRP sequence was inserted into pQE-30-Xa to construct expression vector pQE-30-CIRP.It was transformed into host strain E.coli M15 and was induced by IPTG. It shows efficient expression in the E.coli by SDS-PAGE detected. The expression product exists in inclusion body. CIRP fusion protein was purified by Ni-NTA affinity chromatography and electroelution. The purified CIRP protein was used to immune BALB/C mice three times. Result of indirect ELISA shows that antibody titers of serum was 1∶105. The hybridoma technique was adopted to fusion. Positive hybridoma cell was screened by indirect ELISA and 3 times subcloning. Western blot was used to indentify McAb specificity. Indirect ELISA was used to identify McAb IgG subclasses and determine titer of McAb cell supernatants and ascites. 1A6, 2D3, 3C5 and 4C4 4 hybridoma cell line of stable secretion CIRP McAb was obtained. The antibody subclass of 1A6, 3C5, 4C4 were IgG2b equally, The antibody subclass of 2D3 was IgG3, light chains were κ chain equally. 4 monoclonal cell supernatant titers were more than 1∶103, ascites titer reaching more than 1∶107. Western blot analysis indicates four monoclonal antibodies can specifically combine with recombinant protein CIRP and had no response with void vector comparison. The ascites of cell line 3C5 was as primitive antibody to test natural CIRP expression status of a variety of cold treated organizations. It shows the ascites of cell line 3C5 can specifically recognize natural CIRP protein of heart, liver, spleen, lung, kidney, brain, testes and other tissues, and can be combined with CIRP McAb. This proves that the CIRP McAb has a good specificity and binding activity. McAb against CIRP will act as biological reagents to be used in study of the further research of CIRP functions and utilization.
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Received: 29 October 2009
Published: 25 March 2010
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Corresponding Authors:
shize li
E-mail: lishize1@sina.com
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