Molecular Cloning And Expression Of I-type Lysozyme And Phage-type Like Lysozyme Gene From Freshwater Shellfish

In the study,i-type lysozyme(CpLYZ3)cDNA was cloned from Cristaria plicata,and phage-type lysozyme(HcPLYZ)cDNA was cloned from Hyriopsis cumingii,using rapid amplification of cDNA ends and nested PCR.The full-length cDNA sequence of CpLYZ3 was 968bp,the 5' untranslated region(UTR)and 3' UTR was 395bp and 141bp respectively,and contained a 432bp open reading frame(ORF)coded for a 143-amina acids.The calculated molecular mass and isoelectric point(PI)of deduced protein were 15.6KDa and 6.09,respectively.SignalP program analysis showed that there is a signal peptide contain 17-amino acids in the N-terminus of deduced protein.The multiple sequence alignment analysis fount that this gene has the two highly conserved i-type lysozyme activity sites(Glu61 and Ser72).And the sequence homology analysis found that this gene has 27-87%the high homology with the other i-type lysozyme gene.So we can preliminarily concluded that the CpLYZ3 belongs to the i-type lysozyme.The expression of CpLYZ3 mRNA in Cristaria plicata was measured by fluorescent real-time quantitative RT-PCR.The results showed that the mRNA expression of CpLYZ3 had significant differences in the organization.The expression level of CpLYZ3 in hepatopancreas was the highest,followed by the mantle and gill,the expression level of CpLYZ3 in muscle is the lowest.The expression levels of CpLYZ3 in hemocytes,hepatopancreas and gill were increased significantly after the Aeromonas hydrophila and PGN challenged,and the highest expression levels of CpLYZ3 is in the hepatopancreas.So it show that the lysozyme has the main role in the hepatopancreas.The CpLYZ3 sequence was connect with the expression vector PET-30a(contain the histidine label),and we successful build a prokaryotic expression vector PET30a-LYZ3.The expression vector was transformed into the cells of BL21 and cultured for 8h under the optimal conditons(1mmol/L IPTG,transformation temperature 37?).The recombinant protein was confirmed by SDS-PAGE,and the molecular weight of the recombinant protein was 21 kDa,which was accord with the predicting outcomes.The full-ength cDNA sequence of HcPLYZ was 829bp,consisting of a 5'-terminal untranslated region(UTR)of 97 bp,a 3'-terminal UTR of 264 bp.The open reading frame of 468bp,and encoded a polypeptide of 154 amino acids with a predicted molecular mass of 17.6kDa,and a theoretical isoelectric point of 5.89.The N-terminus had no signal peptide as defined by SignaIP software,and the mature peptide harbored three cysteine residues.The deduced amino sequence of HcPLYZ contains two highly conserved phage-type lysozyme activity sites(Glu20 and Asp29).The microbial from organization in Hyriopsis cumingii was coating on the LB culture medium,and then pick the colony for PCR,we can found that the HcPLYZ was also in the microbial,therefore presumed the HcPLYZ was cloning from the symbiotic bacteria in the Hyriopsis cumingii.The expression of HcPLYZ mRNA in Hyriopsis cumingii was measured by fluorescent real-time quantitative RT-PCR.The results showed that the mRNA expression of HcPLYZ had significant differences in the organization,the expression levels of HcPLYZ in hemocytes,hepatopancreas and gill were increased significantly after the Aeromonas hydrophila challenged,and the expression levels of HcPLYZ in gill were higher than bland control in all time after the Aeromonas hydrophila.In order to further inquire into the functions of HcPLYZ,PET30-PLYZ prokaryotic expression system was constructed by double digestion,and prokaryotic expression plasmid was transformed into Escherichia coli(DE3).The recombinant protein was successfully expressed after IPTG induction,and purified by using the native Ni2+ affinity chromatography.Micrococcus luteus as substrate,the optimum pH of the recombinant HcPLYZ was 5.5,and the optimum temperature was 50?.