[Production of HMGN2 polyclonal antibody by immunization with recombinant GST-HMGN2 fusion protein and its application to analysis of HMGN2 distribution in human monocytes].
OBJECTIVE
To prepare high mobility group chromosomal protein N2 (HMGN2) polyclonal antibodies and determine the subcellular localization of HMGN2 in human monocytes.


METHODS
The recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was constructed and E. coli-based product of GST-HMGN2 fusion protein was prepared and used to immunize rabbit for producing the anti-serum against HMGN2. The polyclonal antibodies were partially purified by caprylic acid and ammonium sulfate precipitation. The titter of specific polyclonal antibodies against HMGN2 was detected by ELISA. The immunocytochemical staining was performed to determine the distribution of HMGN2 in THP-1 cells.


RESULTS
Gel electrophoresis of the enzyme-digested recombinant plasmid and the DNA sequencing confirmed that the recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was correctly constructed. After IPTG induction, the recombinant-transformed E. coli produced a bulk of GST-HMGN2 fusion protein. The polyclonal antibodies to HMGN2 was obtained from the serum of rabbit immunized with GST-HMGN2 fusion protein and its ELISA titer was 1:2000. The immunocytochemistry staining indicated that when stimulated with LPS, HMGN2 was present not only in THP-1 nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant.


CONCLUSIONS
This result suggests that recombinant peptide fusion protein could be used to produce peptide antibody. HMGN2 could be present in the cytoplasm of monocytes and release to the extracellular environment when stimulated with lipopolysaccharide (LPS).
Xiong
Wen-bi
WB
Research Unit of Infection & Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Feng
Yun
Y
Wang
Guo-xing
GX
Huang
Ning
N
Wu
Qi
Q
Li
Xuan
X
Wang
Bo-yao
BY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002460:Cell Line; D024242:HMGN2 Protein / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D024242:HMGN2 Protein; D011993:Recombinant Fusion Proteins