Cloning And Verification Of Lactoferricin B And Several Sequence Mutations In Yeast S.cerevisiae

Objective To construct the eucaryotic recombinant plasmid of pYES2/LactoferricinB and express in yeast of S.cerevisiae and preliminary verify the antibacterial activity.Methods The gene of Lactoferricin B and its several sequence mutations wereamplified from the parts of the pre-synthesized single chains by self-template PCRmethod. They were cloned into the vector of pYES2 to construct the recombinedexpression plasmid pYES2/Lactoferricin B etc. Extracted the purpose plasmid andtransformed them into the yeast of S.cerevisiae. The expressions of proteins weredetermined after induction by galactose. The proteins were collected and purifiedby hydronium-exchange column, and bacteria inhibiting test identified theantibacterial activities.Results The PCR amplifying and DNA sequencing tests indicated the purposeplasmid contained the gene of Lactoferricin B and several mutations. Theinducted proteins were confirmed by SDS-PAGE electrophoresis and massspectrum test. The vito antibacterial activities of the mutations were verifiedpreliminary.Conclusion The recombined plasmid pYES2/Lactoferricin B etc. wereconstructed and inducted expressed in yeast of S.cerevisiae; the recombined protein of Lactoferricin B was obtained, which provides a basis for further research work on it's biological function and antibacterial activity.