Cloning And Expression Of Cathepsin L Proteins Of Giardia Duodenalis And Their Effect On Macrophage Inflammation

Giardia duodenalis is a zoonotic pathogen associated with gastrointestinal diseases.It is widely distributed around the world and about 200 million people are infected with G.duodenalis every year.G.duodenalis has only two stages in its life cycle,including the trophozoite stage,which can lead to disease,and the cyst stage,which can resist the harsh environment for a long time.G.duodenalis trophozoites use abdominal suction cups to adsorb on the mucosa of duodenum and proximal jejunum and proliferate in a dichotomous manner.Human G.duodenalis infections occur mainly via water-borne transmission.Drinking or eating water and food containing cysts can cause G.duodenalis infection.G.duodenalis infection can cause symptoms such as diarrhea,weight loss,and flatulence,as well as various complications such as arthritis,irritable bowel syndrome,and chronic fatigue syndrome.G.duodenalis is the most primitive single-cell eukaryote,and has important research value as a model of eukaryote research.Cathepsin is an important part of the cysteine protease family,widely exists in various animal tissues and cells,and has been widely studied because of its various biological functions.With the development of research,it was found that cathepsins play an important role in various biological processes,such as antigen processing and presentation,apoptosis,tumor progression and metastasis,bone resorption and osteolysis,parasite infection,tiss ue invasion and regeneration,immune escape,etc..Cathepsin L is a member of the cathepsin family,which has strong endopeptidase activity,and its role in degrading proteins and other extracellular components seems more powerful than other proteases.Cathepsin L is mainly involved in protein degradation,antigen processing,antigen presentation,tumor cell invasion,and metastasis in the body.In the researches involving parasites,it was found that cathepsin L mainly plays an important role in parasites' nutrient uptake,cell or tissue invasion,immune escape,individual development,and tissue differentiation.Therefore,the research on cathepsin L of parasitic animals not only helps to reveal the mechanism of interactions between parasitic animals and hosts,but also opens up new ideas and directions for the prevention and treatment of parasitic diseases.In this study,the cathepsin L(CTSL-1,CTSL-2,and CTSL-3)genes of G.duodenalis were selected,and bioinformatics techniques were used to analyze and predict its hydrophilicity,hydrophobicity,signal peptide,structure domain,secondary structure,and tertiary structure.Primers were designed based on the three cathepsin L gene sequences,and the target genes of CTSL-1,CTSL-2,and CTSL-3 were successfully amplified using G.duodenalis genome sequence as a template.We successfully constructed recombinant expression plasmids p Cold-CTSL-1,p Cold-CTSL-2,p Cold-CTSL-3,p Cold-TF-CTSL-1,p Cold-TF-CTSL-2,and p Cold-TF-CTSL-3 using p Cold vector and p Cold-TF vector.The inclusion body recombinant proteins CTSL-1,CTSL-2,and CTSL-3 and soluble recombinant proteins CTSL-TF-1,CTSL-TF-2,and CTSL-TF-3 were successfully expressed and obtained.We used recombinant proteins CTSL-1,CTSL-2,and CTSL-3 to immunize mice three times to prepare polyclonal antibodies.We used Western Blot and enzyme-linked immunosorbent assay to detect and analyze polyclonal antibodies.The results showed that the prepared polyclonal antibodies had good antigen specificity and excellent antigen binding activity.Cathepsin L proteins were widely distributed in the cytoplasms of G.duodenalis trophozoites by the use of indirect immunofluorescence localization technology.We used three different concentrations of soluble recombinant proteins to stimulate macrophages J774 A.1,and then used nitric oxide assay kit to detect the level of nitric oxide in the cell supernatant and used real-time fluorescence quantitative PCR to detect the cytokine IL-1? and TNF-? levels.The results showed that the expression of nitric oxide was increased following G.duodenalis cathepsin L stimulated macrophages,the expression of cytokines IL-1? and TNF-? was increased,and also showed a concentration-dependent effect.The results indicated that CTSL-1,CTSL-2,and CTSL-3 proteins can induce inflammation by stimulating the secretion of nitric oxide and pro-inflammatory factors IL-1? and TNF-? in macrophages.This study attempts to reveal the function of G.duodenalis cathepsin L when G.duodenalis acts on host cells,which will be of great significance for G.duodenalis control and vaccine development.