Molecular Cloning And Expression Analysis Of The Lysozyme Gene CcLys2 From Coridius Chinensis

Lysozyme exists widely in animals,plants and microorganisms,and can selectively decompose the cell wall of microorganisms.For a long time,lysozyme has been used as a"model"system to study the spatial conformation of proteins,enzyme dynamics and the relationship with molecular evolution and molecular immunity.Multicellular animals resist infection by two systems called innate immunity and acquired immunity.However,in the long-term process of biological evolution,insects have formed different immune systems from vertebrates.Insects only have innate immune systems.Insect antimicrobial polypeptides are important members of innate immunity and play an important role in the process of insect resistance to pathogenic microorganisms,among which lysozyme is the most famous.At present,there are two kinds of lysozymes in insects,c-type and i-type.However,most of the lysozymes with functional characteristics at the protein level are insect c-type lysozymes,while i-type lysozymes only have characteristics at the DNA or m RNA level.Coridius chinensis belongs to hemiptera:dinidoridae,which is a commonly used medicinal insect in China.The research indicated that the hemolymph of the insect had broad-spectrum antibacterial activity,which mainly came from various antimicrobial polypeptides in the hemolymph.Therefore,the study of C.chinensis lysozyme is not only of great significance to the innate immune mechanism of C.chinensis,but also lays a theoretical foundation for the further development of C.chinensis resources.In this study,a c-type lysozyme,CcLys2(Gen Bank Number:MN816376),was isolated from the transcriptome data of C.chinensis.The CcLys2gene was cloned and its evolutionary relationship with other insect c-type lysozymes was studied.The expression pattern of CcLys2 gene and the expression levels of CcLys2 after infection with bacteria were studied,and the role of CcLys2 in the growth,development and innate immunity of C.chinensis were discussed.CcLys2was successfully expressed in Escherichia coli BL21(DE3)and Pichia pastoris GS115 expression system,and its structure and antibacterial activity were studied.Results of the study are as follows.1.Cloning and characteristics analysis of CcLys2 geneCcLys2 was derived from the transcriptome data of C.chinensis.Blast results in NCBI database showed that it was a c-type lysozyme gene.The CcLys2 gene sequence was 1096 bp by sequencing,and the open reading frame was 672 bp,encoding 223 amino acids(AAs).The predicted theoretical molecular weight of CcLys2 is 24.8 k Da,and the isoelectric point p I=5.09.The amino acid sequence of CcLys2 is divided into two parts:signal peptide and mature peptide,including a signal peptide cleavage site(A13?K14),13 AAs N-terminal signal peptide and 210 AAs C-terminal mature peptide.Subcellular localization prediction analysis showed that CcLys2 was located outside the cell and belonged to secretory protein.Domain analysis showed that CcLys2 belonged to the c-type lysozyme and?-lactalbumin family.It had a conserved c-type lysozyme domain LYZ1 and eight cysteine residues(C₆-C₁₂₄,C₂₇-C₁₁₃,C₆₂-C₇₃and C₆₉-C₈₇)that may form disulfide bonds.Besides,CcLys2 also have two catalytically essential residues:glutamic acid E32 and aspartic acid D50.2.Spatiotemporal expression profile of CcLys2 geneThe expression pattern of the CcLys2 gene was detected using real-time quantitative PCR(RT-q PCR).The RT-q PCR results showed that the expression levels of the CcLys2 gene were different in various instars.The expression levels of CcLys2in the 2^nd-instar nymph were the highest and significantly higher than that in other instar nymphs,followed by the 5^th-instar nymph.Also,in different tissues of the adult,the results indicated that the expression of CcLys2 was mainly in the fat body,followed by the hemolymph,and also in the midgut.The expression levels of CcLys2in other tissues were low,almost no expression.3.Bacterial induced expression of CcLys2 geneThe expression level of CcLys2 in adults was up-regulated 6 to 60 h after injecting C.chinensis with bacteria,reaching the highest level at 60 h,and then declined.Obviously,CcLys2 is involved in the immune response to bacterial infection.In addition,the expression level of CcLys2 was significantly higher than that of the control group after 6 h by feeding C.chinensis with bacteria,indicating that feeding bacteria can induce CcLys2 and that this enzyme is at least partially secreted in the intestine.4.Expression and activity detection of CcLys2 in E.coliIn order to express CcLys2 in E.coli BL21(DE3)expression system at the best level.According to the codon preference of E.coli,the nucleotide sequence of mature peptide CcLys2 was optimized by using Gen Smar Codon Optimization Tool(beta version 1.0).The restriction endonuclease recognition sites Nde I and Eco R I were added at the 5'and 3'ends of the corresponding gene sequence.The 6×His-tag is added to the N-terminal of the amino acid sequence to facilitate the purification of recombinant lysozyme.The optimized sequence was synthesized,subcloned into PET-28b(+)plasmid and transformed into E.coli.The positive clones were induced by 1 mmol/L IPTG.After the His-tagged fusion protein was purified using Ni-NTA affinity chromatography,SDS-PAGE showed a clear band at about 28 k Da(Figure7b),and Western blot revealed a blotting band at about 28 k Da.After renaturation by dialysis,the purified CcLys2 showed the muramidase activity.CcLys2 has the lytic effect on M.luteus,Bacillus subtilis and Streptococcus agalactiae among the four gram-positive bacteria,but it has the lytic effect on Staphylococcus aureus.In addition,no lytic activity of CcLys2 was observed for the 4 gram-negative bacteria(E.coli,Pseudomonas solanacearum,Pseudomonas aeruginosa and Salmonella typhi)that participated in the test.5.Expression and antibacterial activity of CcLys2 in P.pastorisThe target gene is connected to the recombinant plasmid,linearized and phosphorylated,and then electrotransformed into the Pichia pastoris competent cell GS115,so that the p PICZ?A-CcLys2 plasmid is integrated into the P.pastoris genome.Zeocin was used to screen positive clones and identify the Mut⁺phenotype,and clone and sequence to verify the positive recombinant strain GS115/p PICZ?A-CcLys2.The positive recombinant strain was induced with 0.5%methanol.After purification,the expression product was detected by SDS-PAGE and Western blot,and the specific band and blotting at about 27 k Da was found in the fermentation supernatant.Similar to CcLys2 after prokaryotic expression renaturation,the concentrate of the induced supernatant has lytic activity against Gram-positive bacteria,but the expression of CcLys2 in P.pastoris is small,and it is on the premise of maintaining the activity Purification is relatively difficult.