Preparation And Application Of Polyclonal Antibody Against Human Histone Acetyltransferase HElp3

Elp3, which possesses the activity of the histone acetyltransferase (HAT), is an important subunit of Elongator complex. As a transcription elongation factor, it plays important roles in recruiting chromatin remodeling proteins, and in modulating chromatin structure by acetylating the highly conserved lysine residues in N-terminal tails of histones.In order to study the functions of human Elp3, a fragment of the coding region of hElp3 gene was amplified by PCR from the genomic DNA from HeLa cells. The DNA fragment was inserted into the prokaryotic expression vector pGEX-5X-1 and then transformed into the E.coli BL21 strain to express the 6×His-Elp3 fusion protein after induction by IPTG. The fusion protein thus produced can be readily purified by Ni2+-NTA affinity chromatography. The Elp3 fragment obtained was used as the antigen to immunize a rabbit. In the first immunization, equal volume of Freund's Complete Adjuvant was mixed with antigen solution to enhance the antigenicity. After two more times of strengthening immunization, the serum of the rabbit was sampled and purified as the polyclonal antibody. We determined the concentration of the antibody by ELISA and used it to immunoblot the Elp3 protein for confirmation of its activity. The polyclonal antibody of Elp3 was used in ELISA to investigate the influence of the expression of Elp3 protein in human embryonic kidney cell line 293T transfected by the specific Elp3 antisense oligodeoxynucleotides. The results demonstrate that the expression level of Elp3 was decreased in a dose-dependent manner, suggesting that the polyclonal antibody of Elp3 was effective.The work in this thesis will be beneficial for studying the functions of Elp3 in human cell lines and will contribute to elucidating the mechanisms of histone acetylation in eukaryotic gene transcriptional regulation.