旋毛虫cystatin-like基因重组蛋白诊断特性的鉴定
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摘要
为鉴定旋毛虫肠道期肌幼虫cystatin-like基因重组蛋白的诊断特性,利用ELISA方法,以旋毛虫cystatin-like基因重组蛋白作为包被抗原,并以旋毛虫感染早期肠道6 h幼虫ES抗原作为阳性对照,用不同旋毛虫感染剂量、不同感染时间小鼠血清为一抗,检测血清中抗旋毛虫Ig G抗体的变化情况,利用Excel软件绘制出Ig G抗体消长规律曲线并进行数据分析,鉴定该基因重组蛋白作为诊断抗原时的敏感性及诊断效果。结果显示,在低剂量感染和高剂量感染时,该基因重组抗原分别从感染后第10天和第8天起可以稳定地检测出抗旋毛虫Ig G抗体;在感染后第11天至第45天一直呈"波峰波谷"式并维持较高水平;感染早期(第15~45天)低剂量感染组的Ig G抗体水平明显高于高剂量组。由此推断,旋毛虫cystatin-like基因重组蛋白具有很好的早期诊断特性,尤其对低剂量早期感染的诊断效果更佳,并推测该基因极有可能在旋毛虫感染早期对宿主的免疫抑制作用中起重要作用,其具体应用前景和生物学功能还有待于进一步研究。
To identify the diagnostic characteristics of cystatin-like gene protein of the intestinal stage larvae of Trichinella spiralis,ELISA was used using the cystatin-like gene recombinant protein as envelope antigen and the excretory-secretory(ES) antigen of early intestinal 6 hours muscle larvae(6 h ML) was made as positive control to detect the level of anti-Trichinella antibody Ig G with different infection doses and days post infection(dpi).The antibody Ig G rules were mapped by Microsoft Excel,which indicated the sensitivity and the effects for diagnosis of cystatin-like gene recombinant protein.The results showed that,in all groups,the anti-Trichinella antibody Ig G was positive steadily from the 10 dpi and 8 dpi;the curve of antibody Ig G was "crest wave trough" type and maintained at a high level from 11 dpi to 45 dpi;antibody Ig G level was significantly higher at the early stage(from 15 dpi to45 dpi) in low dose group.Therefore,cystatin-like gene recombinant protein has excellent diagnostic effects for early serodiagnosis,especially in low dose infection,and it is speculated that cystatin-like gene is likely to play an important role in host immune inhibition at a early stage of Trichinella infection.Biology functions of cystatin-like gene recombinant protein remain to be further studied.
To identify the diagnostic characteristics of cystatin-like gene protein of the intestinal stage larvae of Trichinella spiralis,ELISA was used using the cystatin-like gene recombinant protein as envelope antigen and the excretory-secretory(ES) antigen of early intestinal 6 hours muscle larvae(6 h ML) was made as positive control to detect the level of anti-Trichinella antibody Ig G with different infection doses and days post infection(dpi).The antibody Ig G rules were mapped by Microsoft Excel,which indicated the sensitivity and the effects for diagnosis of cystatin-like gene recombinant protein.The results showed that,in all groups,the anti-Trichinella antibody Ig G was positive steadily from the 10 dpi and 8 dpi;the curve of antibody Ig G was "crest wave trough" type and maintained at a high level from 11 dpi to 45 dpi;antibody Ig G level was significantly higher at the early stage(from 15 dpi to45 dpi) in low dose group.Therefore,cystatin-like gene recombinant protein has excellent diagnostic effects for early serodiagnosis,especially in low dose infection,and it is speculated that cystatin-like gene is likely to play an important role in host immune inhibition at a early stage of Trichinella infection.Biology functions of cystatin-like gene recombinant protein remain to be further studied.
引文
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[10]WANG Z Q,SHI Y L,LIU R D,et al.New insights on serodiagnosis of trichinellosis during window period:early diagnostic antigens from Trichinella spiralis intestinal worms[J].Infectious Diseases of Poverty,2017,6(1):41.
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[14]文慧,王李昂,刘春颖,等.旋毛虫病人血清Western blot对成虫可溶性蛋白中早期诊断抗原的鉴定[J].中国病原生物学杂志,2017,12(2):132-135.WEN Hui,WANG Li-ang,LIU Chun-ying,et al.Identification of soluble proteins from adult Trichinella spiralis worms in Western blots of sera from patients with trichinellosis and use of those antigens to diagnose trichinellosis in its early stages[J].Journal of Pathogen Biology,2017,12(2):132-135.(in Chinese)
[15]WANG L,WANG Z Q,HU D D,et al.Proteomic analysis of Trichinella spiralis muscle larval excretory-secretory proteins recognized by early infection sera[J].Bio Med Research International,2013,2013:139745.
[16]WANG L,CUI J,HU D D,et al.Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics[J].Parasites&Vectors,2014,7:40.
[17]黄学贵,贺莉芳,袁仕善,等.旋毛形线虫肌幼虫代谢分泌抗原的鉴定[J].细胞与分子免疫学杂志,2016,32(5):635-639.HUANG Xue-gui,HE Li-fang,YUAN Shi-shan,et al.Identification of serological antigens in excretory-secretory antigens of Trichinella spiralis muscle larvae[J].Chinese Journal of Cellular and Molecular Immunology,2016,32(5):635-639.(in Chinese)
[18]BIEN J,CABAI W,MOSKWA B.Proteomic analysis of potential immunoreactive proteins from muscle larvae and adult worms of Trichinella spiralis in experimentally infected pigs[J].Folia Parasitologica,2015,62(1):22.
[19]CUI J,WANG L,SUN G G,et al.Characterization of a Trichinella spiralis 31 k Da protein and its potential application for the serodiagnosis of trichinellosis[J].Acta Tropica,2015,142:57-63.
[20]SUN G G,LIU R D,WANG Z Q,et al.New diagnostic antigens for early trichinellosis:the excretory-secretory antigens of Trichinella spiralis intestinal infective larvae[J].Parasitology Research,2015,114(12):4637-4644.
[21]SUN G G,WANG Z Q,LIU C Y,et al.Early serodiagnosis of trichinellosis by ELISA using excretory-secretory antigens of Trichinella spiralis adult worms[J].Parasites&Vectors,2015,8:484.
[22]孙召金,刘晓雷,唐斌,等.旋毛虫感染刺激宿主产生抗旋毛虫抗体的消长规律[J].吉林农业大学学报,2015,37(4):477-481.SUN Zhao-jin,LIU Xiao-lei,TANG Bin,et al.Dyna mics of anti-Trichinella antibodies from the stimulated host by T.spiralis[J].Journal of Jilin Agricultural University,2015,37(4):477-481.(in Chinese)
[23]WANG L,TIAN X Y,SUN G G,et al.The use of recombinant 31 k Da antigens of Trichinella spiralis for serodiagnosis of experimental trichinellosis[J].Iranian Journal of Parasitology,2015,10(2):230-237.
[24]BELL R G,MCGREGOR D D,WOAN M C,et al.Trichinella spiralis:selective intestinal immune deviation in the rat[J].Experimental Parasitology,1983,56(1):129-142.
[25]LANGELAAR M,ARANZAMENDI C,FRANSSEN F,et al.Suppression of dendritic cell maturation by Trichinella spiralis excretory/secretory products[J].Parasite Immunology,2009,31(10):641-645.
[2]于申业,吴秀萍,王学林,等.旋毛虫p46000抗原基因的转录及表达特性鉴定[J].中国兽医科学,2009,39(5):389-394.YU Shen-ye,WU Xiu-ping,WANG Xue-lin,et al.Stage specific transcription and expression of p46000antigen gene in Trichinella spiralis[J].Chinese Veterinary Science,2009,39(5):389-394.(in Chinese)
[3]WU X P,FU B Q,WANG X L,et al.Identification of antigenic genes in Trichinella spiralis by immunoscreening of c DNA libraries[J].Veterinary Parasitology,2009,159(3-4):272-275.
[4]吴秀萍,刘相叶,王学林,等.旋毛虫肠道1期幼虫抗原性基因的筛选与分析[J].动物医学进展,2009,30(8):50-54.WU Xiu-ping,LIU Xiang-ye,WANG Xue-lin,et al.Immuno-screening and analysis of antigenic genes from Trichinella spiralis L1 at intestinal stage[J].Progress in Veterinary Medicine,2009,30(8):50-54.(in Chinese)
[5]ROBINSON M W,MASSIE D H,CONNOLLY B.Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis[J].Molecular and Biochemical Parasitology,2007,151(1):9-17.
[6]TANG B,LIU M,WANG L,et al.Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage[J].Parasites&Vectors,2015,8:78.
[7]翟铖铖,陈家旭,陈韶红,等.旋毛虫病诊断方法研究综述[J].中国动物检疫,2017,34(6):68-73.ZHAI Cheng-cheng,CHEN Jia-xu,CHEN Shao-hong,et al.Research progress of diagnosis methods about trichinellosis[J].China Animal Health Inspection,2017,34(6):68-73.(in Chinese)
[8]简莎娜,艾琳,陈韶红,等.旋毛虫病免疫诊断抗原的研究进展[J].中国病原生物学杂志,2015,10(4):384,附页1-2,封三.JIAN Sha-na,AI Lin,CHEN Shao-hong,et al.Research advance on immunodiagnostic antigens for trichinellosis[J].Journal of Pathogen Biology,2015,10(4):384,insert 1-2,inside back cover.(in Chinese)
[9]YANG Y,CAI Y N,TONG M W,et al.Serological tools for detection of Trichinella infection in animals and humans[J].One Health,2016,2:25-30.
[10]WANG Z Q,SHI Y L,LIU R D,et al.New insights on serodiagnosis of trichinellosis during window period:early diagnostic antigens from Trichinella spiralis intestinal worms[J].Infectious Diseases of Poverty,2017,6(1):41.
[11]LIU R D,JIANG P,WEN H,et al.Screening and characterization of early diagnostic antigens in excretory-secretory proteins from Trichinella spiralis intestinal infective larvae by immunoproteomics[J].Parasitology Research,2016,115(2):615-622.
[12]LIU R D,QI X,SUN G G,et al.Proteomic analysis of Trichinella spiralis adult worm excretorysecretory proteins recognized by early infection sera[J].Veterinary Parasitology,2016,231:43-46.
[13]WANG Z Q,LIU R D,SUN G G,et al.Proteomic analysis of Trichinella spiralis adult worm excretorysecretory proteins recognized by sera of patients with early trichinellosis[J].Frontiers in Microbiology,2017,8:986.
[14]文慧,王李昂,刘春颖,等.旋毛虫病人血清Western blot对成虫可溶性蛋白中早期诊断抗原的鉴定[J].中国病原生物学杂志,2017,12(2):132-135.WEN Hui,WANG Li-ang,LIU Chun-ying,et al.Identification of soluble proteins from adult Trichinella spiralis worms in Western blots of sera from patients with trichinellosis and use of those antigens to diagnose trichinellosis in its early stages[J].Journal of Pathogen Biology,2017,12(2):132-135.(in Chinese)
[15]WANG L,WANG Z Q,HU D D,et al.Proteomic analysis of Trichinella spiralis muscle larval excretory-secretory proteins recognized by early infection sera[J].Bio Med Research International,2013,2013:139745.
[16]WANG L,CUI J,HU D D,et al.Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics[J].Parasites&Vectors,2014,7:40.
[17]黄学贵,贺莉芳,袁仕善,等.旋毛形线虫肌幼虫代谢分泌抗原的鉴定[J].细胞与分子免疫学杂志,2016,32(5):635-639.HUANG Xue-gui,HE Li-fang,YUAN Shi-shan,et al.Identification of serological antigens in excretory-secretory antigens of Trichinella spiralis muscle larvae[J].Chinese Journal of Cellular and Molecular Immunology,2016,32(5):635-639.(in Chinese)
[18]BIEN J,CABAI W,MOSKWA B.Proteomic analysis of potential immunoreactive proteins from muscle larvae and adult worms of Trichinella spiralis in experimentally infected pigs[J].Folia Parasitologica,2015,62(1):22.
[19]CUI J,WANG L,SUN G G,et al.Characterization of a Trichinella spiralis 31 k Da protein and its potential application for the serodiagnosis of trichinellosis[J].Acta Tropica,2015,142:57-63.
[20]SUN G G,LIU R D,WANG Z Q,et al.New diagnostic antigens for early trichinellosis:the excretory-secretory antigens of Trichinella spiralis intestinal infective larvae[J].Parasitology Research,2015,114(12):4637-4644.
[21]SUN G G,WANG Z Q,LIU C Y,et al.Early serodiagnosis of trichinellosis by ELISA using excretory-secretory antigens of Trichinella spiralis adult worms[J].Parasites&Vectors,2015,8:484.
[22]孙召金,刘晓雷,唐斌,等.旋毛虫感染刺激宿主产生抗旋毛虫抗体的消长规律[J].吉林农业大学学报,2015,37(4):477-481.SUN Zhao-jin,LIU Xiao-lei,TANG Bin,et al.Dyna mics of anti-Trichinella antibodies from the stimulated host by T.spiralis[J].Journal of Jilin Agricultural University,2015,37(4):477-481.(in Chinese)
[23]WANG L,TIAN X Y,SUN G G,et al.The use of recombinant 31 k Da antigens of Trichinella spiralis for serodiagnosis of experimental trichinellosis[J].Iranian Journal of Parasitology,2015,10(2):230-237.
[24]BELL R G,MCGREGOR D D,WOAN M C,et al.Trichinella spiralis:selective intestinal immune deviation in the rat[J].Experimental Parasitology,1983,56(1):129-142.
[25]LANGELAAR M,ARANZAMENDI C,FRANSSEN F,et al.Suppression of dendritic cell maturation by Trichinella spiralis excretory/secretory products[J].Parasite Immunology,2009,31(10):641-645.