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中国生物工程杂志

China Biotechnology
China Biotechnology  2009, Vol. 29 Issue (03): 30-35    DOI:
    
Prokaryotic expression and Preparation of Polyclonal Antibody of Human NANOGP8
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Abstract  

To obtain polyclonal antibody against human NANOGP8, the NANOGP8 gene was amplified by PCR from the template obtained in our previous work and identified by DNA sequence analysis. Then it was digested by EcoRⅠand SalⅠ, and ligated with pET-28a vector which was by the same treatment. Sequenced and blasted with the NCBI GenBank, the recombinant plasmid was named as pET-28a-NANOGP8. The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by 1mM IPTG at 16℃ and 37℃. SDS-PAGE showed that the fusion protein was expressed in the form of inclusion body in the sediment when induced at 37℃. After dissolution and affinity purification with Chelating Sepharose Fast Flow, a single protein band about 45 KD was identified in the SDS-PAGE and Western-blot analysis. One new zealand rabbit was immunized with purified NANOGP8 protein to prepare for the polyclonal antibody against NANOGP8. The NANOGP8 antiserum was obtained and characterized by Western-blot, ELISA, and compared with commercial goat anti-human Nanog antibody. Results showed that the antibody had high affinity and specificity, and higher titer compared with commercial anti-human Nanog antibody. The studies provide a favorable tool for further study on relation of tumorigenesis, development and NANOGP8 in the future.



Received: 24 October 2008      Published: 31 March 2009
Cite this article:

ZHANG Jing-Tu- Wang-Ai-E- Li-Mei-Xiang- Dai-Jian-Wu. Prokaryotic expression and Preparation of Polyclonal Antibody of Human NANOGP8. China Biotechnology, 2009, 29(03): 30-35.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2009/V29/I03/30

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