Analysis And Expression Of Cathepsin L-transferases In Cyclina Sinensis

Cyclia sinensis is a common benthic shellfish which is both nutritious and delicious,and has important economic and nutritional value.It has become a major shellfish mariculture for its adaptability and disease resistance,while the large-scale death of Cyclina sinensis caused by pathogen infection has been reported frequently as the expansion of farming scale and the continuously improvement of breeding density.So the in-depth study of immune system and disease-related factors of Cyclina sinensis will provide new theoretical guidance for the prevention and treatment of diseases in aquaculture so as to promote the development of aquaculture industry,deepen the understanding of innate immune system of invertebrate,and lay the foundation for further scientific research.Cathepsin L-transferases(CPLs)is a principal member of cysteine proteases C1 family of the papain family.It is widely present among all the various types of biological tissue,but has differences in the expression patterns.The following functions are known to Cathepsin L:protein degradation,some precursor protein(plasminogen and prohormone)hydrolyzation and generation of its active form,activation of other proteolytic enzyme system to make them work and participate in a variety of physiological activities of the body.New researches in medical field shows that Cathepsin L plays an indicative role in a variety of diseases study.In this study,the Cathepsin L-transferases of Cyclina sinensis(CsCPL)were cloned with cDNA library.The activities of Cathepsin L-transferases(CPLs)were tested under Vibrio anguillarum stress,then the expression of CsCPL detected by the real-time quantitative PCR.We used pET30a(+)and BL21(DE3)pLysS to express CsCPL protein and its inhibitor,and detected the biological activity,functioin and interaction of the products,and also explore the potential role of CsCPL in innate immune response process of Cyclina sinensis.Contents are as follows:Firstly,the Cathepsin L-transferases was screened in the cDNA library of Cyclina sinensis.The cDNA sequence is 1632bp,with a 1050bp ORF which encode 349 amino acids,and its theoretical molecular weight was 38.84KDa.It has a conservative N-terminal domain with protein binding sites(G sites),while its C-terminal domain has larger differences and specific substrate binding site(H sites)which determines the specificity of substrate of cathepsin L.There are 4 catalytic sites(Gln149,Cys155,His294,Asn316)and 2 signature sequence(E58X3RX3F/WX2NX3IX3N77,G90X1NX1FX1D96)in the activity catalytic domain.The results of sequence alignment shows that the sequence of papain gene family is highly conserved,as the consistency between CsCPL and cathepsin L3 gene of Cathepsin family in Sinonovacula constricta reaches up to 68%.The phylogenetic tree shows that CsCPL gene is close to cathepsin genes of molluscs in evolutionary distance,far away to the insects and vertebrates.In conclusion,CsCPL can be classified as Cathepsin type L.Secondly,this study focuses on the CsCPL gene expression in Cyclina sinensis by real time PCR,measures the distribution of CsCPL genes in 5 different tissues and analysis its time-dependent expression in the hemolymph under Vibrio anguillarum infection.CsCPL gene expressed in all 5 tissues and shows the highest level in hemolymph which is significantly higher than the others(P<0.05),and the lowest in liver.After infected by the Vibro,the expression level in hemolymph significantly increased in 3h(P<0.05)and reached its highest level at 6h(P<0.01)which was about 2.7 times than the control group.It came to decline after 24h which was still significantly higher than the control group(P<0.01)and finally reduce to the normal level after 48-96h.So we can tell that the Vibro infection has a strong induction on CsCPL gene.Immune factors' related genes start to synthesize a large number of antimicrobial substances after the organism being infected,and the speed of synthesis slows down in the negative feedback mechanism when the pathogen is inhibited.That's why the expression of CsCPL in Cyclina sinensis showing a downward trend after the first rising.Thirdly,the recombinant CsCPL protein was over-expressed by the plasmid pET30a(+)and the bacteria BL21(DE3)pLysS and purified with 6xHis-Tagged Protein Purification Kit.Result showed that the recombinant protein was detected at 1h after the induction of 1mmol/L IPTG.And the optimum inducement time was 3h.The fusion protein molecular weight was about 35KD.The recombinant CsCPL protein was used to do the antibacterial experiments after renaturation,and was found to have obvious antibacterial activity.Fourthly,the enzyme activity of the fusion protein was detected and its physiological function was analyzed.The fusion protein was found to exhibit highest levels of enzymatic activity in about 300C and the pH at 8.2.Experiments of the recombinant protein were taken to probe its antibacterial activity in vitro and immune function in vivo.The results show that,the fusion protein antibacterial action wasn significant,though not as much as the Lysozyme.And when the protein was injected into the clam mixed with the Vibro,the clam showed greater microbial removal efficiency then control group which was just inj ected with the Vibro.It suggested that the fusion protein played an important role of immune regulation of the clam.In conclusion,the study indicate that CsCPL plays an important role in activities against pathogenic microorganisms stress in Cyclina sinensis.This research enriched the physiological function of CPLs,and provides important experimental data to further clarify the regulation mechanism of immune recognition of the path and immune defense in the clam.