The cloning and prokaryotic expression of a novel gene BC-1514 from activated B lymphocyte

Objective The cloning and prokaryotic expression of a novel B lymphocyte activation related gene. Methods The differential display RT PCR(DDRT PCR) technique was applied to analyze the expression difference of mRNA from resting and activated human tonsil B lymphocytes. The positive differential display cDNA fragments identified by Northern blotting were used as a probe to find activated human B lymphocyte cDNA library. The whole coding sequence of positive clone was amplified by PCR and cloned into the pGEX 5X 1 vector. The recombinant plasmid was transformed into E.coli BL 21 and the expression of the fusion protein was induced by IPTG. Results We obtained a novel cDNA clone using EST32, which was mainly expressed in activated B lymphocyte, as a probe to identify the human B cells cDNA library. The positive cDNA clone (named BC 1514) is 1 514 bp in length and contains a open reading frame of 393bp. The fusion protein of BC 1514 was efficiently expressed in E.coli , took about 14.3% of the total bacterial product. The accession number of cDNA 1514 in GenBank is AF304442. Conclusion We obtained a novel activation related gene in human B lymphocyte, of which the coding sequence was efficiently expressed in E.coli BL 21. [