Construction, Expression And Bioactivity Analysis Of A Novel Bovine Lysozyme Gene

In order to construct a novel lysozyme gene to improve its antibacterial activity and develop effective medicine to overcome the increasing infection of animals by drug resistant bacteria, the experiment was carried out to reconstruct the bovine milk lysozyme gene by use of overlapping PCR technique. The enzyme sites of BamHI were introduced to the C-end, BglII, EcoRI and termination code inserted to the N-end of the bovine lysozyme gene. Two protective bases were added to the outside of enzyme sites, and some part of sequences between α-helix or β-fold structure were deleted. The reconstructed novel bovine milk lysozyme cDNAs was introduced with a linker sequence encoding Ser-Gly-Gly amino acid residues. The result showed that the cloned cDNA sequence was 349bp long, coding 108 amino acids, which accorded with the expected size. Then the BLY cDNA fragment was subcloned into prokaryotic expression vector pGEX-4T-1, forming the prokaryotic expression plasmid (pG-BLY). The recombinant plasmid was digested by BamHI and EcoRI, and identify by plasmid PCR. It was showed that the BLY cDNA fragment was successfully inserted into the plasmid in correct orientation. This laid the foundation for further research the conditions of the inducing expression.The recombinant plasmid of pG-BLY was transformed into the BL21(DE3) competent cells, incubated in 2xYT culture. The expression of the fusion protein was induced with IPTG. The protein expression was assayed by SDS-PAGE. The...