Cloning Of LfcinB Gene And Expression Of LfcinB In Escherichia Coli And In Pichia Pastoris

Lactoferrin is an iron-binding glycoprotein. It has many biological functions. It plays an important role in antibiosis,antivirus,immunological regulation etc. Lactoferrin as the most important member of transferring family which modulates the level of freedom ferri ion in animals' body fluid. Native Lactoferrin major exists in animal milk especially in human colostral, and its content in human colostral is 6-8mg/mL. Lactoferricin is a small peptide derived from N-lobe of lactoferrin with acid-pepsin digestion in acidity condition,and its activity of antibacterial is more powerful than lactoferrin. The differences of antibacterial activity from LfcinB,LfcinH,LfcinC,LfcinM were compared from abroad research. The research found that the antibacterial activity of LfcinB is the most powerful. LfcinB as a high-performance and broad-spectrum antibiotic peptide,it displays an enormous potentiality and extensive application perspective in the research of substituting traditional antibiotics. Because of limited resource,setting up an effective expression system in vitro by genetic engineering,which will provide an important basic to gain considerable LfcinB and carry out the further research.This experiment according to the reported sequence of LfcinB gene in GenBank (NO:NM₁80998),synthesized the single strand of LfcinB by chemical method. Took the single strand sequence as template and used the special primer to amplify LfcinB gene. The PCR product was high density and strong specificity, and the length was 92bp approximately. The product of PCR was directly cloned into pMD18-T vector, through the reaction of colony PCR and digestion analysis with BamH I/Sal I enzyme,it could sure preliminary that the purpose gene had cloned into T vector. Sended the bacterium culture which included recombinant plasmid to Shanghai Biotechnology Company for sequencing,and it verified that the homology of LfcinB gene was 100% at nucleotide level between product of PCR and the single strand of synthesizing chemically,so the product amplified was just the LfcinB gene.PCR product was purified and reclaimed,and was digested by EcoR I and Not I restriction enzyme,then reclaimed LfcinB gene for concentration by gel retrieval. The procaryon expression vector pET-22b(+)and the eucaryon expression vector pPIC9K were also digested by EcoR I and Not I restriction enzyme,and then gel reclaimed.The PCR product digested was cloned into pET-22b(+) vector digested and transformed to BL21. Then affixed IPTG to induce expression of recombinant plasmid and collected product of expression induced for 2h,3h,4h. Simultaneously,set up two negative controls. By PCR and digestion analysis of restriction enzyme affirmed that LfcinB gene had been inserted into the multiple clone sites. The expression product was analyzed by SDS-PAGE,and the result showed that the cloned recombinant protein was expressed in the form of inclusion bodies in E.coli cell with molecular weight of 3.2KD approximately,but the controls didn't. Moreover the production induced for 3h was most.The PCR product digested was cloned into pPIC9K vector, and linearized recombinant plasmid with Sal I, and then transformed to GS115 by spHerplast method which gained His⁺ transformants by incubating. Used the YPD plates contain G418 at a final concentration of 0,5, 0.75, 1.0, 1.5mg/mL for screening for multiple copy His⁺ transformants. Then used the multiple copies His⁺ transformants to plate on MM and MD plates for screening for Mut⁺ and Mut^s pHenotype. Then induced the gene to express in Pichia Pastoris. The supernatant collected was dried by lyopHilizer,and then the concentrated product was analyzed by SDS-PAGE. The result of electropHoretic was that in the position of molecular weight of 3.2KD approximately had a griddle of positive product,but the controls didn't have.Which showed that the protein had been expressed. But the expression product could be verified by concentrating,which demonstrated that the production wasn't ideal. The supemant was concentrated as the same way,then detected the antibacterial activity with E.coli and set up controls. The result showed that the control hole in plate didn't have inhibition aperture,but the tentative hole in plate had,and the diameter of hole was approximately 2.4cm,which showed that the LfcinB expressed in Pichia Pastoris had antibacterial activity.