Expression Of Channel Catfish(Ietalurus Punetaus) C-type Lysozyme In Pichia Pastoris And Its Antibacterial Activity

Lysozyme is an antibacterial protein widely distributed in serum,mucus and organs.It is an important component of innate immunity,by catalyzing the bacterial wall of most bacteria to play a bacteriostatic effect.In the process of aquiculture and poultry raising,the use of lysozyme can effectively avoid a substantial accumulation of antibiotics,not to be able to constitute the influence to human body health.The expression of lysozyme in organism is limited,so the method of extraction is very difficult,the cost is too high to synthetic amino acids sequence,with the development of biotechnology,genetic engineering method,has good prospects for industrial expression of high activity of lysozyme in microorganisms.C-type lysozyme(Chicken-type)is come from egg white,which was originally isolated from egg white,the only type exists in the original animal vertebrates and invertebrates,this type is studied widely and thorough in currently.C-type lysozyme has typical structure,namely 8 cysteine residues and 2 enzymatic catalytic sites,four pairs disulfide bonds play an important role for the stable of lysozyme.At present,the study of C-type lysozyme is mainly tissue expression and activity analysis,there are few studies on the use of genetic engineering to achieve the recombinant expression of lysozyme.In this study,the fusion gene of C-type lysozyme of Channel Catfish Channel was designed and reconstructed.The three recombinant genes were respectively: The natural mature peptide gene cflyc of channel catfish.In order to improve the expression level of the target protein,the mature peptide gene was optimized according to the Pichia pastoris preference,and adding His tags in N terminal.in order to improve the protein activity,the N terminal is modified and C terminal is also designed to add His tags and without His tags.In the design stage of the recombinant gene.The prokaryotic plasmid pMD19-T was selected as the vector,the restriction sites Xho I and Xba I were added to 5'and 3' ends of the target gene respectively.The expression vector of pPICZ?A have a shuttle function,At the same time,the pPICZ?A contains ? factor signal peptide,the extracellular secretion of the target protein is more conducive to the collection and purification.Pichia pastoris X-33 was used as the expression strain.The strain contained the alcohol oxidase gene,which could be induced by methanol.The first part,the first unit of reverse transcription cDNA of gill of catfish in the laboratory as template,primers were designed according to the mRNA sequence of NCBI,C-type lysozyme mature peptide gene was obtained from channel catfish,then DNA fragment as template,the signal peptide cleavage sites was added in 5'side of mature peptide gene through PCR,the 6×histidine and the stop codon was added at end of 3'end.The correct pMD19-T-cflyc was double digested after sequencing,it was linked to the expression vector pPICZ?A to construct eukaryotic recombinant expression vector pPICZ?A-cflyc.Recombinant plasmid was linearized using linear restriction enzyme Sac I,which is electrically transferred to Pichia pastoris X-33,by bleomycin resistance and methanol using rapid screening,yeast genomic PCR verification,finally,the recombinant yeast containing the target gene was successfully constructed.Induced by 0.5% methanol,120 hours in the initial expression conditions of pH 6 and 29?,so relatively stable target protein was obtained.The purified samples were obtained by Ni-NTA column immobilized metal ion affinity chromatography.MALDI-TOF-TOF mass spectrometry proved that the purified product is recombinant C-type lysozyme.The method of Folin phenol showed that the expression level of cflyc reached to 2.75mg/L.The agar plate diffusion method and enzyme activity assay showed that the recombinant C-type lysozyme had certain antibacterial activity against Bacillus subtilis.The second part,in order to improve the expression level of target gene,according to the codon bias of Pichia pastoris,the channel catfish C-type lysozyme mature peptide gene optimized and synthesized.Both ends of the optimization and reorganization of lysozyme gene ocflyc was modified,because the first two amino acid is signal peptide cleavage site,so it may be cut off,the nucleotide sequence of two amino acids was removed from the 5' end,6 histidine is added to the 5' end.The expression conditions was optimized under the conditions of 29?,such as different culture time,quantity of methanol,different media,value of pH.Finally,the experimental data showed that BMM medium,1% methanol and 120 hours could achieve the highest expression.Western blot reveal the strip is the target protein,and the further MALDI-TOF-TOF mass spectrometry showed that the purified C-type lysozyme was obtained.It was observed that the expression of 9mg/L was significantly increased from the electrophoresis bands.Bacteriostatic experiments show that the antibacterial effect is not improved,so the need for further recombinant design to improve the activity of the target protein.In the third part,considering the activity of the target protein,on the basis of the second chapter,the target protein gene is further optimized(rocflyc).There are considered in two aspects mainly.On the one hand,the N-terminus of the target sequence was modified by Lys-Arg residues into Lys-Val residues.On the other hand,the 6×his at the C-terminus may interfere with the three-dimensional structure and stability of the target protein,which may have a significant effect on the bacteriostatic activity of the target protein.The one group,histidine tag is added at the C-terminus,the other group is not added.The results showed that the yeast supernatant of without histidine addition had a good inhibitory effect on Listeria monocytogenes.But the addition of histidine tags did not has activity.The experimental results show that the target protein of add the histidine tag has a certain activity.The construction of yeast expression system has a very successful extracellular secretion expression of channel catfish C-type lysozyme without histidine tag,which can be used as green food preservative refrigeration.