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Recombinant Expression Of Marine Organism Lysozyme In Pichia Pastoris And Products Analysis

Posted on:2013-10-14Degree:MasterType:Thesis
Country:ChinaCandidate:G H LuanFull Text:PDF
GTID:2180330467987370Subject:Fermentation engineering
Abstract/Summary:PDF Full Text Request
Lysozyme is a kind of unspecific immunal factor, and it has the function of antibacterial, anti-inflammatory and anti-viral. Therefore, lysozyme could be made into medicine and preservatives, which could partly instead of antibiotics and chemical food preservatives. Marine organism lysozymes were expressed in Pichia pastoris. They are Crassostrea gigas Lysozyme(CgLys) and Stichopus japonicus Lysozyme(SjLys)Firstly, according to the published marine organism lysozyme cDNA sequences of marine organism lysozymes in GenBank, the specific primers were designed and synthesized. Total RNA was prepared from the tissue of marine organism to be used as templates for amplification of marine CgLys and SjLys gene by RT-PCR, respectively. Secondly, the target genes were inserted into multiple cloning site of pPIC9K to construct the recombinant plasmids, and then transformed into the Pichia pastoris GS115by electroporation. The phenotype His+Muts were screened out by MD and MM plate, and the high-copy positive strains were screened out by YPD plate which containing antibiotics G418. At last, the genetic engineering stains of CgLys and SjLys with antibacterial activity were screened by methanol-induced. Two Pichia pastoris GS115expressing strains GS115/pPIC9K-CgLys-31and GS115/pPIC9K-CgLys-42were obtained. Three expressing strains GS115/pPIC9K-SjLys-20, GS115/pPIC9K-SjLys-66and GS115/pPIC9K-SjLys-67were obtained. And both of GS115/pPIC9K-CgLys-31and GS115/pPIC9K-SjLys-66was showen as only one band at14.3kDa on SDS-PAGE; while in the control experiment of GS115/pPIC9K, there was no visible band.CgLys and SjLys were successfully expressed in P. pastoris. Both of them have the antibacterial activity of Vibrio parahemolytious, Staphylococcu saureus, Micrococcus lysodeikticus and Pseudomonas aeruginosa. This research makes academic technological bases for industrial scale production of lysozyme. It will provide a good method to drive the development of scale producing lysozyme.
Keywords/Search Tags:lysozyme, Pichia pastoris, Recombinant expression, Crassostrea gigas, Stichopus japonicus
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