Eukaryotic Expression Of DCD-1L And Plasmid Construction Of Its Random Mutations

Dermcidin is a new antibacterial peptide identified most recently from human sweat glands ,and the gene of DCD-1L is a derivant of Dermcidin which has a broad spectrum of activities against Gram negative and positive bacteria .They are new class of antibacterial medicines having enormous potentialities. Therefore it has great economic value and theoretical significance.In this research we obtain DCD-1L by overlap extention,clone and express it in Pichia pastoris GS115,determine the antifungal activity simply,construct the expression vector of random mutant population of DCD-1L, and try to screen high expressing Pichia recombinant clones.The method is as follow:(1)DNA fragments were designed according to the amino acid sequence and differences in codon usage,obtain DCD-1L by overlap extention and cloned into expression vector pPICZ?-A, Which containsα-factor signal sequence. The recombinant plasmid was sequenced , digested with SacⅠand transformed Pichia pastoris GS115 by electroporation. Screen and determine the Zeocin-resistant colonies. Under the control of the promoter AOX1(alcohol oxidase 1), the peptide was expressed in super-natant protein. Analyze the supernatants by antibacterial activity assay and Coomassie-stained SDS-PAGE.(2)Obtain DCD-1L with he sequence of random mutations by overlap extention and cloned into expression vector pGAPZα-C, Which contains GAP promotor andα-factor signal sequence.The recombinant plasmid was digested with BspHⅠand transformed Pichia pastoris SMDII68 by electroporation.Screen the high expressing Pichia Strains with replica plating from random mutant population of DCD-1L. The results are as follows:1.The DCD-1L and its mutant gene were obtained.2.The DCD-1L gene was correctly inserted into the vevtor pPICZα-A.Sequencing analysis indicated that DCD-1L was identical to the sequence wanted.3. Transformed the recombinant plasmid into Pichia pastoris GS115 by electroporation,the protein band about 6KD appeared on SDS-PAGE gel.4. Through defermination of inhibiting bacteria circle, we observed the inhibition of bacteria with supernatants. 5. The murant of DCD-1L gene was inserted into the vevtor pGAPZα-C. Transformed the recombinant plasmid into Pichia pastoris SMD1168 by electroporation. Research on screening high expressing Pichia Strains is an ongoing process.With this method the desired DCD-1L gene were constructed and expressd in Pichia successfully,and the expression vector of random mutant population of DCD-1L were constructed, it might provide a foundation to screen high expressing Pichia recombinant clones.