Prokaryotic expression and purification of bovine neutrophil β-defensin 5 of Horqin dairy cattle

Objective The express the bovine neutrophil β-defensin 5(BNBD5)in prokaryotic cells and purify the expressed product. Methods Total RNA was extracted from the neutrophils of bovine peripheral blood with Trizol reagent,from which the cDNA sequence of BNBD5 was amplified by RT-PCR and inserted into vector pMD18-T. The constructed recombinant cloning plasmid pMD18-T-BNBD5 was sequenced,and analyzed for homologies of BNBD5 nucleotide sequence and relationships to those of other animals such as sheep,goat,reindeer,house mouse,humans,mandarin fish and Chinese bee. Meanwhile,the BNBD5 nucleotide sequences of Horqin dairy cattle to the cattle of other species, including Xinjiang Holstein cattle,Indian buffalo and Netherlands Friesland cattle,were compared. The constructed recombinant cloning plasmid pMD18-T-BNBD5 was subcloned to prokaryotic expression vector pET-30a(+),and the obtained recombinant plasmid pET-30a-BNBD5 was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by nickel ion affinity chromatography and identified by SDS-PAGE. Results BNBD5 cDNA sequence at a full length of 195 bp was amplified by PCR,which was a intact open read frame and showed a homology of 93. 4% to the sequence of encoding region of BNBD5 reported in GenBank(AJ278799). It was deduced to encode 45 amino acid residues,which contained the characteristic molecular structure of defensin,i.e. six conversed cysteine residues at certain sites. Mutations were observed in 13 bases,resulting 3 variations in the deduced amino acid sequence. However,no variations were observed in the 6 conversed cysteine residues. The homology of BNBD5 nucleotide sequence of Horqin dairy cattle was more than 80% to those of goat and sheep,while was less than 10% to that of Chinese bee. However,the homology was more than 90% to that of Xinjiang Holstein cattle and about 66% to that of Netherlands Friesland cattle. Both PCR and restriction analysis proved that the recombinant plasmid pET-30aBNBD5 was constructed correctly. The expressed protein,with a relative molecular mass of about 10 000,mainly existed in a soluble form,contained 25. 1% of total somatic protein and reached a content of 2. 15 mg / ml after purification. Conclusion The BNBD5 of Horqin dairy cattle was successfully expressed and purified.