犬恶丝虫丝氨酸蛋白酶抑制剂特性分析与诊断价值的初步评价
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摘要
为探究犬恶丝虫丝氨酸蛋白酶抑制剂(Di-Serpin)的生物学特性,初步评价重组蛋白(rDi-Serpin)的诊断价值。本研究扩增犬恶丝虫的Di-Serpin基因片段并经原核表达系统表达该蛋白,经western blot和免疫荧光定位分析r Di-Serpin的免疫反应性以及Di-Serpin在虫体的分布;以纯化的r Di-Serpin为包被抗原,通过间接ELISA评价其诊断价值。结果显示,Di-Serpin基因片段共编码121个氨基酸,预测蛋白质分子量为13.56 ku,等电点为5.06,含有一个Serpin结构域;Di-Serpin主要分布于犬恶丝虫(雌虫、雄虫)的表皮层和肠上皮;以其为包被抗原建立的间接ELISA方法,结果显示:该方法的敏感性和特异性检测结果分别为70%(14/20)和70%(35/50)。研究结果表明,犬恶丝虫的Di-Serpin可能为分泌蛋白,在抑制宿主蛋白酶活性,参与抗炎及逃避宿主对虫体的免疫中起重要作用;该蛋白虽有较好的免疫反应性,但其特异性和敏感性均较低,不适合作为犬恶丝虫病的候选诊断抗原。
In order to investigate the biological characteristics of the Serpin protein from Dirofilaria immitis(Di-Serpin),and evaluate the diagnostic value of the recombinant Di-Serpin(rDi-Serpin),the Di-Serpin gene was amplified,and rD i-Serpin was expressed and purified in this study.The immunoreactivity of rD i-Serpin and distribution of Di-Serpin was analyzed by western blot and immunofluorescence localization.The rD i-Serpin-based indirect ELISA was performed to evaluate the diagnostic value of rD i-Serpin.The results showed that the Di-Serpin gene encoded of 121 amino acids,with a predicted molecular weight of 13.56ku and PI of 5.06,and a serine protease inhibitor domain.Immunofluorescence localization showed that the Di-Serpin mainly distributed in the epidermis and intestinal epithelial of D.immitis(female,male).The sensitivity and specificity of the indirect ELISA method were 70%(14/20)and 70%(35/50),respectively.In this study,the Di-Serpin may be a secreted protein,and it may play an important role in inhibiting protease and escaping the immunity of host proteases by D.immitis.The prokaryotic expression of rD i-Serpin had a good immunoreactivity,but the sensitivity and specificity of the indirect ELISA method were low,which suggest that the rD i-Serpin was not suitable as a diagnostic antigen for D.immitis.
In order to investigate the biological characteristics of the Serpin protein from Dirofilaria immitis(Di-Serpin),and evaluate the diagnostic value of the recombinant Di-Serpin(rDi-Serpin),the Di-Serpin gene was amplified,and rD i-Serpin was expressed and purified in this study.The immunoreactivity of rD i-Serpin and distribution of Di-Serpin was analyzed by western blot and immunofluorescence localization.The rD i-Serpin-based indirect ELISA was performed to evaluate the diagnostic value of rD i-Serpin.The results showed that the Di-Serpin gene encoded of 121 amino acids,with a predicted molecular weight of 13.56ku and PI of 5.06,and a serine protease inhibitor domain.Immunofluorescence localization showed that the Di-Serpin mainly distributed in the epidermis and intestinal epithelial of D.immitis(female,male).The sensitivity and specificity of the indirect ELISA method were 70%(14/20)and 70%(35/50),respectively.In this study,the Di-Serpin may be a secreted protein,and it may play an important role in inhibiting protease and escaping the immunity of host proteases by D.immitis.The prokaryotic expression of rD i-Serpin had a good immunoreactivity,but the sensitivity and specificity of the indirect ELISA method were low,which suggest that the rD i-Serpin was not suitable as a diagnostic antigen for D.immitis.
引文
[1]罗洪林,王豪举,谌剑波.犬恶丝虫硫氧还蛋白过氧化物酶真核表达质粒的构建及初步免疫试验[J].中国预防兽医学报,2013,35(4):325-329.
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[4]盖文燕,王君玮,付宝权.寄生性蠕虫丝氨酸蛋白酶抑制剂研究进展[J].中国动物检疫,2015,(3):57-61.
[5]Nelson C T,Mccall J W,Rubin S B,et al.Guidelines for the diagnosis,prevention and management of heartworm(Dirofilaria immitis)infection in dogs[J].Vet Parasitol,2005,133(2-3):267-275.
[6]Rojas A,Rojas D,Montenegro V M,et al.Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR,blood-concentrating techniques and a serological assay in dogs from Costa Rica[J].Parasit Vectors,2015,8(1):170-180.
[7]Ranjbarbahadori S,Eslami A,Bokaic S.Evaluation of different methods for diagnosis of Dirofilaria immitis[J].Pak J Biol Sci,2007,10(11):1938-1940.
[8]Lee A C Y,Bowman D D,Lucio-Forster A,et al.Evaluation of a new in-clinic method for the detection of canine heartworm antigen[J].Vet Parasitol,2011,177(3-4):387-391.
[9]Courtney C H,Cornell J A.Evaluation of heartworm immunodiagnostic tests[J].J Am Vet Med Assoc,1990,197(6):724-729.
[10]刘德昊,杨光友,王成东,等.犬恶丝虫抗体dot-ELISA检测方法的建立[J].中国兽医科学,2008,38(2):106-110.
[11]Tritten L,Clarke D,Timmins S,et al.Dirofilaria immitis,exhibits sex-and stage-specific differences in excretory/secretory miRNA and protein profiles[J].Vet Parasitol,2016,232(232):1-7.
[12]Solgi R,Sadjjadi S M,Mohebali M,et al.Development of new recombinant DgK antigen for diagnosis of Dirofilaria immitis infections in dogs using ELISA technique and its comparison to molecular methods[J].Iran Biomed J,2017,22(4):283-289.
[13]Ford L,Guiliano D B,Oksov Y,et a1.Characterization of a novel filarial serine protease inhibitor,Ov-SPI-1,from Oncho- cerca volvulus,with potential multifunctional roles duringdevelopment of the parasite[J].J Biol Chem,2005,280(49):40845-40856.
[14]Yang Ya-bo,Hu Dong,Wang Le-xun,et al.Comparison of two serpins of Clonorchis sinensis by bioinformatics,expression,and localization in metacercaria[J].Pathog Glob Health,2014,108(4):179-185.
[15]Sasaki M,Sako Y.The putative serine protease inhibitor(serpin)genes encoded on Echinococcus multilocularis genome and their expressions in metacestodal stage[J].Vet Parasitol,2017,233(233):20-24.
[16]Gettins P G W.Serpin structure,mechanism,and function[J].Chem Inform,2003,34(7):4751-4804.
[17]Little S E,Raymond M R,Thomas J E,et al.Heat treatment prior to testing allows detection of antigen of Dirofilaria immitisin feline serum[J].Parasit Vectors,2014,7(1):1-4.
[18]Velasquez L,Blagburn B L,Duncan-Decoq R,et al.Increased prevalence of Dirofilaria immitis antigen in canine samples after heat treatment[J].Vet Parasitol,2014,206(1-2):67-70.
[2]McC all J W,Genchi C,Kramer L H,et al.Heartworm disease in animals and humans[J].Adv Parasitol,2008,66:193-285.
[3]Whisstock J C,Silverman G A,Bird P I,et al.Serpins flex their muscleⅡ.Structural insights into target peptidase recognition,polymerization,and transport functions[J].J Biol Chem,2010,285(32):24307-24312.
[4]盖文燕,王君玮,付宝权.寄生性蠕虫丝氨酸蛋白酶抑制剂研究进展[J].中国动物检疫,2015,(3):57-61.
[5]Nelson C T,Mccall J W,Rubin S B,et al.Guidelines for the diagnosis,prevention and management of heartworm(Dirofilaria immitis)infection in dogs[J].Vet Parasitol,2005,133(2-3):267-275.
[6]Rojas A,Rojas D,Montenegro V M,et al.Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR,blood-concentrating techniques and a serological assay in dogs from Costa Rica[J].Parasit Vectors,2015,8(1):170-180.
[7]Ranjbarbahadori S,Eslami A,Bokaic S.Evaluation of different methods for diagnosis of Dirofilaria immitis[J].Pak J Biol Sci,2007,10(11):1938-1940.
[8]Lee A C Y,Bowman D D,Lucio-Forster A,et al.Evaluation of a new in-clinic method for the detection of canine heartworm antigen[J].Vet Parasitol,2011,177(3-4):387-391.
[9]Courtney C H,Cornell J A.Evaluation of heartworm immunodiagnostic tests[J].J Am Vet Med Assoc,1990,197(6):724-729.
[10]刘德昊,杨光友,王成东,等.犬恶丝虫抗体dot-ELISA检测方法的建立[J].中国兽医科学,2008,38(2):106-110.
[11]Tritten L,Clarke D,Timmins S,et al.Dirofilaria immitis,exhibits sex-and stage-specific differences in excretory/secretory miRNA and protein profiles[J].Vet Parasitol,2016,232(232):1-7.
[12]Solgi R,Sadjjadi S M,Mohebali M,et al.Development of new recombinant DgK antigen for diagnosis of Dirofilaria immitis infections in dogs using ELISA technique and its comparison to molecular methods[J].Iran Biomed J,2017,22(4):283-289.
[13]Ford L,Guiliano D B,Oksov Y,et a1.Characterization of a novel filarial serine protease inhibitor,Ov-SPI-1,from Oncho- cerca volvulus,with potential multifunctional roles duringdevelopment of the parasite[J].J Biol Chem,2005,280(49):40845-40856.
[14]Yang Ya-bo,Hu Dong,Wang Le-xun,et al.Comparison of two serpins of Clonorchis sinensis by bioinformatics,expression,and localization in metacercaria[J].Pathog Glob Health,2014,108(4):179-185.
[15]Sasaki M,Sako Y.The putative serine protease inhibitor(serpin)genes encoded on Echinococcus multilocularis genome and their expressions in metacestodal stage[J].Vet Parasitol,2017,233(233):20-24.
[16]Gettins P G W.Serpin structure,mechanism,and function[J].Chem Inform,2003,34(7):4751-4804.
[17]Little S E,Raymond M R,Thomas J E,et al.Heat treatment prior to testing allows detection of antigen of Dirofilaria immitisin feline serum[J].Parasit Vectors,2014,7(1):1-4.
[18]Velasquez L,Blagburn B L,Duncan-Decoq R,et al.Increased prevalence of Dirofilaria immitis antigen in canine samples after heat treatment[J].Vet Parasitol,2014,206(1-2):67-70.