Screening Of I-type Lysozyme Produced By Recombinant Pichia Pastoris And Sea Cucumber Feeding Test

Lysozyme is an alkaline protease,it can destroy the ?-1,4 glycosidic bond of the bacteria cell wall and make the bacteria peptidoglycan cell wall "scattered".Further research found that,i-type lysozyme(SjLys)has a broad antibacterial spectrum.and a wider range of potential application.(1)In this study,we first studied the efficient expression of Sj Lys.Three Pichia pastoris genetic strains were used which the lysozyme genes have been optimized according to yeast cell gene preference previously.They are the genetic strain No.1(pPIC9K-SjLys/GS115),the genetic strain No.2(pPIC9K-SjLys-HisN/GS115)and the genetic strain No.3(pPIC9K-opt-SjLys/GS115).The aim is to screen out the high-level expressing strains.First of all,three plasmids of pPIC9K-SjLys(Genetic strain No.1),pPIC9K-SjLys-N(Genetic strain No.2)and pPIC9K-opt-SjLys(Genetic strain No.3)were isolated and then linearized with Bgl?.The plasmids were transferred to Pichia pastoris GS115 by electroporation,then the high copy transformants were further screened by G418 resistance.The high-yield strain was screened by SDS-PAGE gel electrophoresis,and its activity was further verified by the inhibition zone.A high-yield strain was picked and named as HS1-1.(2)To investigate the effects of SjLys on the growth and immunity of sea cucumber,we used the SjLys to feed sea cucumber.Experimental group 1:feeding feed;control group 2:feeding feed and probiotics;experimental group:set the five lysozyme gradient(1000?5000U/kg).The results showed that the activities of superoxide dismutase,alkaline phosphatase,acid phosphatase and lysozyme were compared and analyzed.The results showed that the experimental group with 4000U/kg lysozyme had the best growth performance,the largest increase in body weight and the highest relative immunase activity.Through the experiment,the experimental results showed that the optimal concentration of lysozyme and probiotics was 4000U/kg and 1×108cfu/g.And when the feeding time was 45 days,the activity of enzymes related to immunity and antioxidant in sea cucumbers was the highest,and then all related enzymes remained the trend of stability.The research of this project not only provides a strong material basis for further fermentation experiment of lysozyme,but also provides a theoretical basis and experimental basis for the preparation of sea cucumber i-type lysozyme,which has furture research value.