Construction And Expression Of Lactoferricin And Its Mutant

OBJECTIVE: To obtain normal LfcinB, and make a mutation, express both the normal and the mutant gene in E.coli, determine the normal's antibacterial activity simply.METHODS: According to the sequence of the mature LfcinB cDNA in Genebank, the primers was designed with the restriction endonucleases site of EcoRI and SaiI at 5'end. Construct the mutant according to the reference at the 4th codon for arginine and the 10 for methionine.The normal and the mutant gene were ligated with pUC-18, transformed into the E.coli DH5α, and through a complementary, the positive recombinant was identified and sequenced. The positive recombinant and pGEX-4T-l were digested by EcoRI and SaiI, ligated at 16℃, transformed into E.coli BL21, and then sequenced after the recombinant was identified. The positive recombinant was induced by IPTG for 2~5 hours at 37℃, the results were observed by SDS-PAGE. After harvesting and sonicating the positive E.coli BL21, the inclusion body was obtained, and then was solved, cleaved by thrombin, purified through Glutathione-Sepharase 4B column. Finally, the antibacterial activity of the normal protein was determined by antibacterial assay.RESULTS:1. A fragment about 90bp (including restriction endonucleases site) appeared after sequencing analysis indicated that the mature LfcinB cDNA was identical to the sequence wanted.2. The mutation was made to the mature LfcinB cDNA according to the reference at the 4th codon for and the 10 for. Sequencing analysis indicated that the codon CGT for arginine at position 4th and the ATG for methionine at position 10th were both changed into the codon TGG for tryptophan.3. The LfcinB and the mLfcinB were ligated with pGEX-4T-l expression vector respectively, transformed into E.coli BL21, the recombinant were extracted and identified. Sequencing analysis indicated that the two ligation were correct and could induce the protein expression.4. With IPTG inducing expression for 2~5 hours, the fusion protein band about 29KD appeared on SDS-PAGE gel. After harvesting and sonicating the positive E.coli BL21, the inclusion body was obtained. After the inclusion body was solved, denatured, renatured and purified, the fusion protein was soluble.5. The soluble fusion protein was cleaved by thrombin, and purified through Glutathione- Sepharase 4B column. The antibacterial assay indicated that the LfcinB protein have the antibacterial activity against S.aureus ATCC 25983.CONCLUSION? The LfcinB> mLfcinB gene were constructed and expresseded in E.coli BL21 successfully. After purified the LfcinB showed the antibacterial activity. It might provide a foundation for construction eukaryotic expression vector and research of its biological activity.