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Objective To investigate the long-term toxicity of anti-inflammatory and anticoagulant tick anticoagulant peptide (TAP)-staphylococcal superantigen like protein-5 (SSL5) fusion protein in normal rats.MethodsSD rats were intraperitoneally injected with TAP-SSL5 (1mg/kg, every other day) for eight weeks and followed up for one week.The general behavior, weight, blood routine test, blood biochemistry and organ indexe were measured.ResultsOur results showed that there were no significantly difference between the TAP-SSL5 treated rats and the control on general behavior, weight, blood routine test, blood biochemistry, organ indexe and pathology.ConclusionThe fusion protein TAP-SSL5 with little long-term toxicity for rats is proved to be a safe drug.
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Objective: To investigate the effect of fusion protein tick anticoagulant peptide (TAP)-staphylococcus aureus superantigen-like protein 5 (SSL5) on the formation of atherosclerotic plaque in ApoE knockout (ApoE-/-) mice.Methods: Totally 21 male 12-week-old ApoE-/-mice were randomly divided into three groups: TAP-SSL5 (3 mg·kg-1·d-1) group, SSL5 (2 mg·kg-1·d-1) group and the blank control group (pH 7.4 phosphate buffer), ip, qd, for 12 weeks.The changes of body mass were observed.The mice were fed with high cholesterol diet for 12 weeks, and then the levels of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in plasma were detected.The aorta of mice was subjected to paraffin section and routine HE staining.The formation of atherosclerotic plaque in the aortic root was analyzed.The distribution of atherosclerotic plaques was observed by oil red O staining of the aorta.Results: Compared with that of the blank control group, the increasement of body weight of TAP-SSL5 group and the level of TC significantly decreased (P <0.001), while TG, HDL-C and LDL-C did not change significantly.The HE staining results showed that the plaque area of root slice in the aorta in TAP-SSL5 group was significantly lower than that in the blank control group (P<0.05).The red O staining of aorta showed that the formation of atherosclerotic plaque in TAP-SSL5 group was significantly smaller than that in the blank control group.Conclusion: TAP-SSL5 can significantly inhibit the formation of atherosclerotic plaques in the arteries of ApoE-/-mice.
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Objective: To study the preparation and separation method for anticoagulant peptide and the effect of anticoagulation and thrombolysis in vitro. Methods: The casein was hydrolyzed to prepare anticoagulant peptide using the mixed four enzymes such as papain, pineapple proteinase, neutral protease, and alkali protease. The anticoagulant peptide was extracted using immobilized thrombin. The effect of haemolysis and anticoagulation in vitro was investigated through the New Zealand rabbits experiments. Results: The conditions of preparation anticoagulant peptide were as follows: quality of casein was 15%, papain proteinase, pineapple proteinase, neutral protease, and alcalase dosage were 1500, 2400, 1000, and 1250 U/(g casein), respectively, temperature was 50℃, pH value was 7.0, and hydrolysis time was 4 h. The conditions for the extraction of anticoagulant peptide were as follows: the initial concentration was 6 ATU (Anti Thrombin Unit)/mL, temperature was 30℃, pH value was 5.0, and time was 30 min. Anti-extraction temperature was 30℃, pH value was 7.6, and time was 40 min. The purified anticoagulant peptide was analyzed via high performance size exclusion chromatography. The molecular weight of purified anticoagulant peptide was equal to N-Hippuryl-His-Leu hydrate and the main components were three peptides. The time of anticoagulation was more than 72 h and the time of hemolysis was 24 h in vitro. Conclusion: The main components of anticoagulant peptides are three peptides. The effect of hemolysis and anticoagulation in vitro is good.
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AIM: To study the effect of tick anticoagulant peptide-staphylococcal superantigen like protein 5 (TAP-SSL5), an anti-inflammatory and anticoagulant fusion protein , on the binding of activated platelets to human lym-phocytes.METHODS:Human periphery lymphocytes were isolated by magnetic activated cell sorting (MACS).The toxic-ity of TAP-SSL5 on the viability of Jurkat cell was assessed by CCK-8 assay.Flow cytometry was applied to detect the ex-pression of CD162 (PSGL-1) on the Jurkat cells (human peripheral blood leukemia T lymphocyte cell line ) and the inhibi-tory effect of TAP-SSL5 on the binding of mouse anti-human CD162 monoclonal antibody (KPL-1) to Jurkat cells.Platelets were activated by ADP at concentration of 20μmol/L, the binding rates of activated platelets to Jurkat cells or human lym-phocytes were assayed by flow cytometry .RESULTS:The concentration of TAP-SSL5 below 30 mg/L didn’ t affect the vi-ability of Jurkat cells .TAP-SSL5 at 10 mg/L competitively inhibited KPL-1 binding to Jurkat cells .The binding rates of activated platelets to Jurkat cells or lymphocytes were (11.86 ±4.49)% and (8.32 ±1.00)%, respectively, which de-creased to (6.73 ±2.71)%and (5.51 ±0.70)%after the Jurkat cells and lymphocytes were pre-incubated with 10 mg/L TAP-SSL5 (P <0.05).CONCLUSION:TAP-SSL5 binds to PSGL-1 expressed on lymphocyte surface and directly in-hibits the binding of activated platelets to human lymphocytes , which may be one of the anti-inflammatory mechanisms of TAP-SSL5.
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Objective: To acquire recombinant nematode anticoagulant peptide ( NAP) with high anticoagulant activity. Methods: Pichia pastoris GS115 strain was transformed with recombinant yeast expression vector pPICS. 5K-rNAP. Expression of rNAP was induced with methanol after the identification of positive strains. NAP expressed in the collected yeast culture supernatant was confirmed with SDS-PAGE and Western blot. The biological activity of the products was validated with PT (prothrombin time) , INR (international normalized ratio) and APTT (activated partial thromboplastin time) , respectively. Results: The yeast strains expressing NAP were identified. The rNAP was secreted into culture supernatant with a molecular weight of about 10 kDa due to glycosylation, which is a little bigger than that predicted (8.7kDa). The anticoagulant efficiency of rNAP was confirmed with the in vitro assays. Conclusion: The recombinant nematode anticoagulant peptide with high biological activity was successfully expressed in Pichia pastoris and can be used in the future development of novel anticoagulant agent.