Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of C19orf18 Protein

doi:10.13560/j.cnki.biotech.bull.1985.2016-1031

Abstract

Abstract: The purpose of this study is to clone the human C19orf18 gene,construct the prokaryotic expression system of C19orf18,and prepare the recombinant human C19orf18 protein and its corresponding rabbit polyclonal antibody. According to the sequence reported in GenBank database,we used PCR to amplify intracellular and extracellular segments,and ligated them by flexible peptide,and inserted the target segment into prokaryotic expression vectors pET28a and pET32a. Then the vectors of high expression were selected by inducing a little expression of target protein. Further the highly expressing vector was employed for expressing the great amount of target protein. The C19orf18 protein was purified by Ni-NTA affinity column,and then identified by SDS-PAGE. Following that,New Zealand white rabbits were immunized with the purified proteins four times to produce polyclonal antibody. Specificity of the product polyclonal antibody was affirmed by Western blotting. Our results showed that pET28a was a better vector than pET32a for the expression of C19orf18 protein. More importantly,C19orf18 protein with truncated amino acids was expressed in prokaryotic cells,and its corresponding rabbit polyclonal antibody with high specificity was prepared successfully.

Key words: C19orf18 protein, prokaryotic expression, antigen affinity purification

TONG Xiao-Xia, YANG Yuan-Qin, CHEN Mian, WANG Huai-Yuan, HU Hai-Jun, ZHANG Kang-Jian. Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of C19orf18 Protein[J]. Biotechnology Bulletin, 2017, 33(5): 176-182.

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