Facile synthesis of insulin fusion derivatives through sortase A ligation

Insulin derivatives such as insulin detemir and insulin degludec are U.S. Food and Drug Administration (FDA)-approved long-acting insulin currently used by millions of people with diabetes. These derivatives are modified in C-terminal B29 lysine to retain insulin bioactivity. New and efficient methods for facile synthesis of insulin derivatives may lead to new discovery of therapeutic insulin. Herein, we report a new method using sortase A (SrtA)-mediated ligation for the synthesis of insulin derivatives with high efficiency and functional group tolerance in the C-terminal B chain. This new insulin molecule (Ins-SA) with an SrtA-recognizing motif can be conjugated to diverse groups with N-terminal oligoglycines to generate new insulin derivatives. We further demonstrated that a new insulin derivative synthesized by this SrtA-mediated ligation shows strong cellular and

Screening and Identification of the Peptides Specifically Binding to Human Non-small Cell Lung Cancer NCI-H1299 Cells / 中国肺癌杂志

BACKGROUND@#Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, and one of the malignant tumor with the highest mortality. As the main part of the optical molecular imaging probe, peptide can realize the early screening and diagnosis of tumor and improve the survival rate of patients. The aim of this study was to screen the small-molecule peptide that highly binds to NSCLC NCI-H1299 cells using in vivo phage display technology and to identify their binding specificity by in vitro experiment.@*METHODS@#To prepare a tumor-bearing nude mouse model of NCI-H1299 cells, after 3 rounds of in vivo screening with Ph.D.-C7CTM Peptide Library, phage clones were randomly picked, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) to identify the affinity of phage clones to NCI-H1299 cells. The positive monoclonal phages DNA was extracted and sequenced to obtain the amino acid sequence of the peptides. The peptides with the highest repetition rate was chemically synthesized and labeled with fluorescein (FITC) to prepare optical molecular probe. We preliminary identified the specificity of the probe binding to lung cancer cells by in vitro experiment.@*RESULTS@#After three rounds of in vivo screening, the phages enrichment rate was 341.3 times compared with the first round. Immunohistochemical staining showed that with the increase of screening times, the phages binding to tumor tissues continued to increase, and the binding amount was significantly higher than normal tissues; ELISA results showed that 20 clones among the 30 randomly selected phage clones were positive. After sequencing, the peptide with the highest repetition rate was synthesized and named NSP1; Methyl thiazolyl tetrazolium assay (MTT) and would healing assay showed that NSP1 will not affect cell proliferation and migration. Flow cytometry and immunofluorescence showed specific binding of NSP1 to NCI-H1299 cells.@*CONCLUSIONS@#We successfully obtained the peptide NSP1 that specifically binds to lung cancer NCI-H1299 cells by in vivo phage display, which provide a theoretical basis for NSCLC early diagnosis and targeted therapy.

Screening specific binding peptides of Mycobacterium tuberculosis PPE17 protein by Phage display technique / 公共卫生与预防医学

Objective The specific binding peptide of Mycobacterium tuberculosis PPE17 protein was screened by phage display technique. Methods PPE17 gene was amplified from Mycobacterium tuberculosis genome, cloned into pET28a, expressed in E. coli BL21, purified by Ni2+ column, and identified by SDS-PAGE and Western blot. The purified PPE17 protein was coated into an ELISA plate and screened by phage 7 peptide library. After three rounds of panning, phage plaques were randomly selected for sequencing. DNAMAN was used to analyze and compare the amino acid sequences of the polypeptide encoded by the positive clones. Results PPE17 gene was successfully constructed and expressed, and soluble protein with molecular weight of about 37kD was obtained. From the third round of eluents, 20 plaque were randomly selected. The sequencing results could be translated into 8 polypeptide molecules, among which the polypeptide sequence repeated for 6 times was LKWGHVY. Conclusion The specific binding peptide of PPE17 protein is screened by phage display technology, which is expected to be a small molecular diagnostic reagent for the identification of this antigen.

The inhibition of accessory gene regulator C specific binding peptides on biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride in vitro / 中国修复重建外科杂志

Objective: To investigate the effect of accessory gene regulator C (agr C) specific binding peptides (named N1) on the biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride (PVC) materials in vitro. Methods: Firstly, the two strains (ATCC35984, ATCC12228) were cultured with N1 at concentrations of 100, 200, 400, 800, and 1 600 μg/mL, respectively. The control group was cultured with agrC specific binding unrelated peptides (named N0) at the same concentrations and the absorbance ( A) value was measured after 24 hours to determine the optimal bacteriostatic concentration of N1. The two strains were cultured with N1 and N0 of the optimal concentration, respectively. The A values were measured at 6, 12, 18, 24, 30, and 48 hours to observe the effect of N1 on the biofilm formation ability of Staphylococcus epidermidis. On this basis, the surface structure of the biofilm on the surface of PVC material was observed by scanning electron microscopy after 6, 12, 18, 24, and 30 hours of incubation with PVC material sheet. The thickness of the biofilm was observed by laser confocal microscopy after 6, 12, 18, and 24 hours of incubation with ATCC35984 strain. Results: The optimal bacteriostatic concentration of N1 was 800 μg/mL. ATCC 12228 strain did not form obvious biofilm after being cultured with N1 and N0. When ATCC35984 strain was cultured with N1 and N0 for 12 hours, the difference in biofilm formation ability between groups N1 and N0 was statistically significant ( P0.05). Scanning electron microscopy examination showed that mature biofilm structure was observed in ATCC35984 strain and was not observed in ATCC12228 strain. Laser confocal microscopy observation showed that the number of bacteria in the group N1 was significantly lower than that in the group N0 at 12 hours, and the most of bacteria were dead bacteria. There was no significant difference in the number of bacteria at 6, 18, and 24 hours, and the most of them were live bacteria. The biofilm thickness of group N1 was significantly lower than that of group N0 at 12 and 18 hours ( P<0.05). Conclusion: The intensity of N1 inhibiting the formation of Staphylococcus epidermidis biofilm is dose-dependent. During the aggregation period, N1 can inhibit the biofilm formation by hindering the bacterial growth and aggregation. The inhibition effect on mature biofilm is not obvious.

The effect of pH and ligand density on the endocytosis and exocytosis process of FcBP decorated PEG-PCL micelles on Caco-2 cells / 药学学报

The difference in pH between apical and basolateral side of intestinal epithelial and pH dependence character of the combination of FcRn (neonatal Fc receptor) and ligand might improve the delivery of hydrophobic drugs by facilitating the transcytosis of nanocarriers. Here we designed FcBP (IgG Fc domain-binding peptides) decorated coumarin 6 (C6) loaded poly(ethyl ethylene phosphate)-co-poly(ε-caprolactone) (PEG-PCL) micelles with different ligand densities to study the effect of pH and ligand density on the endocytosis and exocytosis process of micelles on human colon adenanocaricinoma cell lines (Caco-2). Active micelles with different ligand densities and passive micelles were prepared using the thin-film hydration method. The size of the micelles was characterized by dynamic light scattering analysis and the morphology was observed by transmission electron microscope. The endocytosis and exocytosis of the micelles at pH 7.4 and pH 6.0, as well as the effect of FcRn on the endocytosis, were investigated by flow cytometry. The results showed that the size of micelles was about 30 nm, which was not affected by FcBP decoration. We found that pH and ligand density could both influence the endocytosis. The uptake of active micelles was higher at pH 6.0 than at pH 7.4, and an optimal ligand density of endocytosis was appeared in both pH environment. Then we proved that FcBP decorated micelles could be endocytosed at pH 6.0 and exocytosed at pH 7.4, and the exocytosis process was also related to ligand density. Micelles with 10% ligand density had the largest exocytosis, showing the potentiality to deliver drugs through the intestinal epithelial. In addition, the competitive inhibition experiments illustrated that the interaction between FcRn and FcBP were essential to endocytosis. The results will enhance the understanding on the FcBP decorated PEG-PCL micelles for transmemberane drug delivery.

Study on the Anti-Endotoxin Activity of Human Endotoxin Binding Peptide P1 and P2 / 现代检验医学杂志

Objective To study the antiendotoxin activity of P1 and P2 based on the lipopolysacchride binding protein.Method P1 and P2 were designed and obtained.In vitro test,peripheral blood mononuclear cell(PBMC)were extracted from volun-teer 100ml venous blood,the experiment group was arranged as:control group,positive control group,LPS group,LPS+P1 (2 mg/L,5 mg/L,12.5 mg/L)group and LPS+P2(2 mg/L,5 mg/L,12.5 mg/L)group,TNF-α and IL-6 of supernatant liquor in every group were detectd by ELISA.In vivo,40 kunming rice were randomly divided into four groups with ten rice each group:control group,model group,LPS+P1 and LPS+P2 group,Pathologic changes of lung and liver tissues were ob-served by hematoxylin and eosin(HE)staining.Results The serum level of IL-6 and TNF-αin 12.5 mg/L P1 and 2 mg/L, 5 mg/L,12.5 mg/L P2 treatment group were lower than that in model group,the difference was statistically significant(all P<0.01).Serum level of TNF-αor IL-6 in 12.5 mg/L P2 treatment group were similar to that in PMB treatment group, and there was no statistically significant difference(P>0.05).Histologymorphology finding showed that central veins of liver and hepatic sinusoid congestion,hepatic cellular edema existed,occasionally,acidophilic change and spotty necrosis were found,pulmonary interstitial edema,focal hemorrhage,alveolar space stenosis existed.As regards 10 mg/kg P1 treatment group mice,hepatic cellular edema and pulmonary interstitial edema ameliorated.About 10 mg/kg P2 treatment group mice, veins of liver and hepatic sinusoid congestion obviously ameliorated,mild pulmonary interstitial edema exsited.Conclusion The results indicated P1 and P2 had antiendotoxin effect,in vivo and vitro,for 12.5 mg/L P2,its inhibition effect for TNF-αor IL-6 release was positive.

Effects of non-T cell binding peptide(FNS007) on collagen Ⅱ-induced arthritis rat models / 中国免疫学杂志

Objective:To observe the effects of FNS007 on collagen Ⅱ-induced arthritis(CIA) rat models and investigate the underlying mechanism.Methods: CIA model was induced by intradermal injection of Freunds adjuvant and bovine CⅡ.Rats were randomly divided into six groups:normal control group,model group,methotrexate group,high,middle and low doses of FNS007 groups,with 12 rats in each group.FNS007 was gived by intravenous injection,the normal control and model group were administrated with PBS.Observing the paw thickness,ankle joint width and the arthritis scores in the CIA rats during the experiment.On d 22 after injection of the drug, all rats were killed.Interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-6 (IL-6) and level of anti-CⅡantibody in serum were examined by enzyme-linked immunosorbent assay (ELISA).The pathological score and radiography of ankle joint were evaluated.Results: Data revealed that FNS007 treated groups showed a significant reduction in paw thickness,ankle joint width and the arthritis scores compared to model group (P<0.05,P<0.01),especially FNS007 high dose goup.The levels of TNF-α,IFN-γ,IL-6 and anti-CⅡantibodies in serum in high dose goup were significantly lower than those of model group(P<0.05,P<0.01).X-ray examination showed that FNS007 could significantly alleviate the damage of joint and decrease the radiographic scores.Pathological examination exhibited that FNS007 could significantly reduce pathological scores,alleviate inflammatory cell infiltration and synovial hyperplasia,improve the histopathological changes.Conclusion: FNS007 has a treating effect on CIA rats,and the mechanisms may be through competitive inhibition of T cell,inhibiting inflammatory cytokines and anti-CⅡantibodies secretion,regulating the abnormal immune responses.

Effects of the synthetic peptide coatings inhibiting the growth of Streptococcus gordonii on titanium surface / 天津医药

Objective To detect the independently designed synthetic peptide adsorbed to the titanium surface and its inhibitory effect on streptococcus gordonii, and to provide a new means for antibiosis reseach on oral implants. Methods The physical and chemical properties of the synthetic peptide and antimicrobial peptide were measured by ExPASy Prot?Param tool, ProtScale analysis, circular dichroism and Zeta potential instrument. The synthetic peptide was anchored on the surface of the titanium specimen through incubation at room temperature. The adsorption of the synthetic peptide to the titani?um surface was examined by X-ray photoelectron spectroscopy (XPS) and the atomic force microscope (AFM). The inhibitory effect on streptococcus gordonii of the synthetic peptide fixed on the titanium surface was viewed by confocal laser scanning microscopy (CLSM). The destructive effects of the synthetic peptide and the antimicrobial peptide on streptococcus gordonii were observed through the transmission electron microscope (TEM). Results The independently designed synthetic peptide still had the physical and chemical properties that the antimicrobial peptide desired. The synthetic peptide had already been detected on the titanium surface after incubated in a 5 g/L synthetic peptide solution. The titanium specimen fixed with the synthetic peptide inhibited the survival and adhesion of streptococcus gordonii. Conclusion It suggests that the indepen?dently designed synthetic peptide might have reached the goal of bacterial inhibition on the titanium surface.

Selection and identification of H1N1 influenza virus hemagglutinin inhibitory peptides / 国际药学研究杂志

Objective Using the influenza virus hemagglutinin (HA) as the target to screen for novel anti-influenza polypeptide drugs. Methods The HA binding peptides were screened out through affinity selection from a 12-peptide phage library, and the anti-H1N1 activity was evaluated at MDCK cell and chicken embryo(ovo). Results Nine HA binding peptides were finally obtained, and the H6 peptide was found having significant antiviral activity against H1N1. Its IC50 against two strains of H1N1, A/FM1/1/47 (H1N1) and A/PPR8/34(H 1 N 1), were 37. 3 and 48. 5 μmoZL respectively determined by cytopathic effect (CPE)test, and 26. 7 and 33. 4 μmoI/L respectively measured by ovo antiviral experiment. Conclusion These results showed that H6 might be a potential herapeu icdrug for H1N1 infecion.

Selection of peptides bind to PreS1 antigen from a phage-displayed peptide library / 第二军医大学学报

Objective: To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library. Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph. D. -7 peptide library with phage display technique, and the positive clones were identified by ELISA. Results: After three rounds of biopanning, the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA. Sequencing result showed that the binding peptides had high affinity and specificity. Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library, which paves a way for the diagnosis and treatment of hepatitis B infection.

Estudo clínico da aplicação de matriz inorgânica de osso associado a peptídeo sintético de adesão celular (MIO/P-15), PepGen P-15®, em lesões periodontais avançadas de cães / Clinical study of effectiveness of an anorganic bone matrix and cell-binding peptide (ABM/P-15), PepGen-P15®: application in advanced periodontal lesions of dogs

Os mecanismos biológicos desenvolvidos para aumentar a qualidade da regeneração óssea e da reparação tecidual de sítios periodontais específicos continuam a ser um desafio e têm sido complementado pela capacidade de adesão celular do colágeno do tipo I, promovida por um peptídeo sintético de adesão celular (P-15), associado a uma matriz inorgânica de osso (MIO) para formar MIO/P-15. O objetivo deste estudo foi avaliar a perda do nível clínico de inserção e a resposta da bolsa periodontal em dentes após 3 e 6 meses da aplicação de enxerto com MIO/P-15. Vinte e um cães do Hospital Veterinário da Universidade de São Paulo foram anestesiados para realização de tratamento periodontal e 132 faces dentais com perda de nível clínico de inserção foram tratadas, sendo que 36,4 por cento (48 faces) receberam o peptídeo de adesão celular e 63,6 por cento (84 faces) compuseram o grupo controle que recebeu tratamento convencional (retalho muco-gengival e aplainamento radicular). O procedimento foi documentado através de radiografia intra-oral e todas as sondagens de bolsas periodontais foram fotografadas. Depois de 3 e de 6 meses, os animais foram re-anestesiados a fim de se obter novas avaliações, radiografias, fotografias e sondagens periodontais. As 48 faces com perda de nível clínico de inserção que receberam material de enxertia apresentaram taxa de 40 por cento de recuperação do nível clínico de inserção após 6 meses. O grupo controle de faces dentais não apresentou alteração do nível clínico de inserção. A face palatina foi a que apresentou melhor taxa de regeneração (40 por cento) e os dentes caninos e molares mostraram as melhores respostas (57,14 por cento e 65 por cento, respectivamente). Não houve sinais de infecção pós-cirúrgica relacionadas à falta de higienização oral dos animais. Pode-se concluir que o MIO/P-15 auxilia na regeneração e re-aderência das estruturas periodontais, incluindo osso alveolar. Sua aplicação mostrou-se fácil...

The development of biologic modalities designed to enhance bone regeneration and wound healing of specific periodontal sites continues to be a challenge and has been accomplished through the cell binding activity of Type-I collagen. These have been provided by a synthetic cell biding peptide (P-15), associated to a anorganic bone matrix (ABM) to form ABM/P-15. The aim of this study was to evaluate the attachment loss and periodontal pocket response in teeth after 3 and 6 months with ABM/P-15 graft application. Twenty one dogs from the Veterinary Hospital, University of São Paulo, were anesthetized in order to accomplish periodontal treatment and 132 teeth faces with attachment loss were treated. From these, 36.4 percent (48 faces) received cell binding peptide and 63.6 percent (84 faces) compounded the control group that received conventional treatment (muco-gingival flap and root planning). The procedure was documented by intra-oral radiography and all periodontal probings were photographed. After 3 and 6 months, the animals were re-anesthetized in order to accomplish new photography, radiography and periodontal probing exams. The 48 attachment loss faces that received graft material exhibited 40 percent of regeneration rate after 6 months. The control faces did not change their attachment level. The palatal face presented the better regeneration rates (40 percent) and the canines and molars teeth showed the better responses (57.14 percent and 65 percent, respectively). There was no post-surgical infection related to absence of oral home care. It can be concluded that ABM/P-15 helps a more rapidly periodontal structure re-attachment and regeneration, including alveolar bone. Its application was easy and practical, and the post-surgical complications incidence was low. Nevertheless, more work is necessary to evaluate the amount and the quality of formed bone and periodontal ligament.

Resistin Binding Peptide Stimulates Basal Insulin Secretion of RINm5F Insulinoma Cells / 实用儿科临床杂志

Objective A resistin binding peptide (RBP) was selected by phage display in our previous work. Studies had shown that RBP could antagonize the role of resistin on the lipid metabolism and endocrine function of adipose tissue, but whether RBP affects the insulin secretion of pancreatic cells is still unknown. The aim of this study is to assess the effect of RBP on basal insulin secretion in RINm5F insulinoma cells. Methods The cell viability was measured by 3-[4,5-dimethyhhiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay. The supernatants were assayed for insulin content by enzyme linked immunosorbent assay (ELISA). Reverse transcriptase-PCR assay and Western blotting were used to determine the expression of glucose transporter 2 (GLUT2) involved in insulin secretion. Cytosolic Ca2+, the trigger of insulin exocytosis, was analyzed with the fluorescent probe FURA-3/AM. Results RBP did no effect on the cell viability with a concentration of 10-8-10-12mol/L of 2 hours intervention. But it stimulated basal insulin secretion of RINm5F cells, accompanied by up-regulated increased expression of GLUT2 and elevated concentration of cytosolic Ca2+. Conclusion RBP could stimulate basal insulin secretion without affecting the cell viability.

Construction of the fusion protein between EGFP and IgG-binding peptide / 中国海洋药物

Objective Making the fusion protein of IgG-binding peptide with enhanced green fluorescent protein(EGFP) and determining its bioactivity.Methods The enhanced green fluorescent protein(EGFP) gene was cloned into pEZZ 18 vector containing ZZ peptide gene to construct expression vector pSpA-EGFP-His.The fusion protein was expressed in E.coliDH5? and its bioactivity was detected by competitive ELISA and fluorescence properties.Results The fusion protein migrated at approximately 42kD in SDS-PAGE,which correspond to the theoretical molecular weight.The spectra of SpA-EGFP fusion protein was similar to what was reported.SpA-EGFP competed with SpA-Peroxidase to bind IgG.Conclusion The plasmid pSpA-EGFP-His correctly expressed in E.coli.The fusion protein retains the bifunctional effects of EGFP and IgG-binding activity.

Selection of peptides bind to PreS1 antigen from a phage-displayed peptide library / 第二军医大学学报

Objective:To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library.Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph.D.-7 peptide library with phage display technique,and the positive clones were identified by ELISA.Results: After three rounds of biopanning,the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA.Sequencing result showed that the binding peptides had high affinity and specificity.Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library,which paves a way for the diagnosis and treatment of hepatitis B infection.

The effect of the resistin-binding peptide (RBP) on the 3T3-L1 adipocyte differentiation,lipid metabolism and GLUT-4 gene expression / 中国糖尿病杂志

Objective To investigate the effect of RBP on 3T3-L1 adipocyte differentiation,lipid metabolism and glucose transporter 4(GLUT-4)gene expression.Methods We constructed an expression vector for rat resistin gene and transfected it into 3T3-L1 adipocytes.RBP was added to the medium of 3T3-L1 adipocytes or resistin-overexpressing adipocytes on day 0 of differentiation.Cell differentiation and lipid accumulation were determined by oil red O staining.The mRNA expressions of differentiation marker genes(pref-1,C/EBP?,FAS)and GLUT-4 gene were evaluated by RT-PCR.Triglyceride(TG)and free fatty acids(FFAs)in adipocytes were measured by colorimetric kit.Results(1)When 10-12mol/L RBP was applied,the percent of living cells was high and the shape was unchanged.(2)RBP had no effect on the differentiation of normal adipocytes,but significantly decreased the number of lipid droplets in resistin-overexpressing adipocytes without affecting the lipid droplets-presenting day.(3)C/EBP? and FAS expressions in resistin-overexpressing adipocytes were down-regulated after RBP was applied,without changing their expressions in normal adipocytes.(4)RBP had no effect on the cellular TG and FFAs levels in normal cells,whereas it can significantly decrease the levels in resistin-overexpressing adipocytes.(5)There was no difference in the expression of GLUT-4 gene between 3T3-L1 adipocytes and RBP-applied cells.Conclusions(1)RBP has no effect on the cell differentiation and lipid metabolism in normal 3T3-L1 adipocytes.(2)RBP can inhibit the cell differentiation and lipid metabolism of resistin-overexpressing 3T3-L1 cells.(3)RBP has no effect on the expression of GLUT-4 gene.

Selection of Cd~(2+) Binding-peptides and Their Affinities for Heavy Metal Ions / 微生物学通报

Selection and affinities of Cd binding peptides were assayed by phage random dodecapeptide library and affinity chromatography. Two Cd binding peptides were obtained, it was found that the affinities of Cu~ 2+ ,Co~ 2+ ,Zn~ 2+ ,Ni~ 2+ for Cd binding peptides were higher than that of Cd~ 2+ and Cr~ 2+ after detection of the amplified Cd binding peptides displayed phages to different heavy metal-charged resins; the detoxification of E.coli to Ni~ 2+ and Cd~ 2+ was enhanced when infected by Cd binding peptide displayed phages as compared with those of the control. The interaction of Cd binding peptide displayed phages with heavy metals charged resins was also observed under microscope. The work would be of great value and consequences for the study of interaction between heavy metals and proteins(peptides), as well as thedetoxification and bioremediation of heavy metals.

Roles of endotoxin binding peptide in rat model of burn combined with endotoxemia / 第三军医大学学报

Objective To observe the protective effect of recombinant human endotoxin binding peptide (EBP) on a rat model of burn combined with endotoxemia. Methods A total of 78 rats of model of burn combined with endotoxemia were divided into three groups: model group ( n =36), treatment group ( n =36), and control group ( n =6). Rats in the model group were intraperitoneally injected with 1 mg/kg endotoxin (prepared with 1 ml saline) immediately after burn. After intraperitoneal injection of 1 mg/kg endotoxin (prepared with 0.5 ml saline), rats in the treatment group were intraperitoneally injected with EBP (prepared with 0.5 ml saline). Blood and liver and lung tissues of rats in the model and treatment groups were collected at 1, 3, 6, 12, 24, and 48 h after treatment. Rats in the control group were intraperitoneally injected with 1 ml saline after trichomadesis. Blood and liver and lung tissues of rats in the control group were collected for the total control. The changes of alanine aminotransferase (ALT), TNF ?, and IL 6 contents in serum were determined by biochemical velocity analysis, ELISA, and light microscopy. The pathological changes of the liver and lung tissues were observed light microscopically and electron microscopically. Results The serum TNF ? level of rats in the model group was significantly higher than that in the control at 1 h after injury ( P

Screening and function analysis of targeted short binding peptides of Endoglin / 重庆医科大学学报

Objective:To do screening in phage displayed 12-peptide library to seek short peptides capable of binding Endoglin detect their affinity constants.Methods:After three rounds of screening,16 phage clones were randomly selected and their affinity was identified by sandwich ELISA.The DNA sequence of positive phage clones was determined,and their speciality was identified by competitive inhibition test.We finally calculated out the affinity constants of those positive phage binding peptides by non-competitive ELISA method.Result:After three rounds of screening,the enriched phage clones were identified. 6 of 16 phage clones were identified positive by competitive ELISA,which had comparatively strong binding activity to rhEndoglin.Five sequences were obtained,and the predominant sequence was AHKHVHHVPVRL.Competitive inhibition test showed that positive phage clones had good affinity to Endoglin,with the affinity constant as(1.431?0.293)?107 mol/L.Conclusion:The rhEndoglin-binding peptides with high affinity to Endoglin can be obtained through the screening of phage random peptide library, which is beneficial to the further studies on the function of Endoglin in the early diagnosis of ovarian cancer.