Expression And Characterization Of Chicken Granzymes As Well As Granulysin

Granulysin?GNLY?and granzyme?Gzm?are important effectors for innate immune secreted by cytotoxic T lymphocypes?CTLs?and natural killer?NK?cells.As described previously,human GNLY can be existed in tissues as two forms of molecular weight,i.e.9 and 15 kD.9-kD granulysin is a matured effector molecule and can directly effect on bacteria,protozoa,infected host cells and tumor cells.Granzymes,members of serine proteases family,can enter the pathogen-hosted target cells and tumor cells in collaboration with GNLY and/or perforin,and induce caspase-independent/dependent apoptosis mechanisms to kill/eliminate pathogens and tumor cells.In this study,the genes for coding cicken GNLY and Gzms were hitted through searching the chicken genome database.In order to probe the effects/mechanism of ChGNLY and/or ChGzms on E.tenella,we cloned and expressed the ChGNLY,ChGzmA and ChGzmK genes,and analyzed their biochemical characterization by enzymological and cytoxic methods.The main research contents and results of these studies include:1.The cloned sequences coding matured ChGzmA and ChGzmK using RT-RCR are 783 bp and 789bp in length respectively.After identification by DNA sequencing,ChGzmA and ChGzmK were inserted into pET26b for constructing the recombinant expressing plasmids pET26b-ChGzmA and pET26b-ChGzmK and fusion proteins were expressed under introduction of TPTG in E.coli BL21?DE3?.The activity of recombinant ChGzmA and ChGzmK was evaluated by enzyme kinetics methods.In our results,the character parameters against Suc-VANR-pNA in ChGzmA are K_m=0.0250±0.00086?M,V_max=122.96±8.49?M/min/?g;for Ac-YRFK-pNA in ChGzmK are K_m=0.0083±0.00061?M,V_max=22.225±5.3832?M/min/?g respectively.D-Phe-Pro-Arg-CMK can inhibit their activity and the character parameters against D-Phe-Pro-Arg-CMK in ChGzmA and ChGzmK are IC₅₀=16.62±1.0629?M and 25.464±1.7731?M respectively.2.The dynamic transcriptions of four chicken granzymes?ChGzmA,ChGzmK,ChGzmM and ChGzmG-like?of cecal tonsils after inoculation were profiled using real-time quantitative PCR method.The results revealed that there was no direct relationships or differences in the mRNA transcription level between infected and uninfected chicken.3.The sequence fragment for coding the matured region of ChGNLY was cloned using RT-RCR based on the SMART online package.The cloned coding sequence of ChGNLY is 240 bp in length and was inserted into pGEX-6p-1 for expressing recombinant ChGNLY by IPTG inducing in E.coli.The rChGNLY can directly lyze the cultured MDBK cells with a dose-dependent effect by CellTiter-Glo^?2.0 Assay and may have potential efficacious actions to the pathogens such as Eimeria spp as predicted by functional motif,activity-essential amino acid and surface potential of rChGNLY molecule.These primary results have fundamental supports for the further researches on the lytic effects and mechanism of ChGNLY and/or ChGzms on Eimeria spp.