旋毛虫肌幼虫特异性诊断抗原的研究
|
摘要
旋毛虫病(trichinellosis)是一种严重的人兽共患寄生虫病，主要因生食或半生食含有旋毛虫幼虫囊包的猪肉或其他动物肉类所致。目前全世界大约有1100万人体旋毛虫感染者，国际旋毛虫病委员会(International Commission on Trichinellosis，ICT)在1995-1997年间报道1万多例病人。我国自1964年发现人体旋毛虫病以来，已在12个省市区发生548起人体旋毛虫病暴发。河南省为全国旋毛虫病的高发区。本病不仅严重危害人体健康，还可造成巨大的经济损失。但目前对本病尚无满意的诊断方法，确诊本病主要靠肌肉活检，但在轻度感染者和感染早期往往不易检出，且不易被患者接受。因此，国内外学者对旋毛虫病的免疫学诊断进行了大量研究，但所用的诊断抗原多为旋毛虫成囊期(肌肉期)幼虫虫体粗抗原，由于抗原成份复杂，用于本病患者的免疫学诊断时常与其它寄生虫病(如并殖吸虫病、华支睾吸虫病、日本血吸虫病及囊虫病等)有交叉反应而出现假阳性，不能取得特异性诊断。为了寻找诊断旋毛虫病的特异性抗原，本研究首先应用SDS-PAGE对旋毛虫肌幼虫可溶性抗原、肌幼虫分泌-排泄(ES)抗原组分进行了分析，然后用旋毛虫病和其它寄生虫病患者血清及正常人和小鼠血清进行免疫印迹(Western blot)。此外，为了鉴定出引起交叉反应的抗原，还对卫氏并殖吸虫及华支睾吸虫成虫可溶性抗原组分进行了分析。
将旋毛虫肌幼虫、卫氏并殖吸虫成虫和华支睾吸虫成虫分别研磨、超声破碎、冷浸、反复冻融、离心，制备虫体可溶性抗原；将旋毛虫肌幼虫在无血清的1640培养基中于5％的CO_2培养箱中分别培养18、21、24、30小时，分别离心、收集上清、透析，低温快速冰冻浓缩后得到培养18、21、24及30小时的旋毛虫肌幼虫ES抗原。测定各种抗原的蛋白含量。将上述各种抗原分别进行SDS-PAGE分析，电泳时用11％分离胶和4％积层胶。充分染色、脱色后可看到各自不同的电泳带。根据标准分子量蛋白带的迁移率及其分子量的对数绘制标准曲线，测量各条蛋白带迁移率，查出与其对应的分子量。然
后将各种抗原用Westem blot分析。SDS一PAGE电泳及Westem blot结果如下。
1.电泳样本蛋白含量的选择:加样孔内蛋白含量的不同，电泳带清晰度有所差异;
样品浓度越高，加样量越少，电泳结果越好;用Bi。一Rad公司的Mini电泳系统时，加
样量在14林岁孔左右;用国产的电泳系统时，加样量在25“g/孔左右。加样量在加样孔
高的1/4处时电泳效果最好。
2.肌幼虫可溶性抗原分析:旋毛虫肌幼虫可溶性抗原有29条蛋白带，主要分子
量范围在66切a~3 IkDa、20kDa~12切a之间，主要蛋白带为65、43、42、31、30、20、
17、16kDa。
3.培养不同时间的肌幼虫ES抗原分析:旋毛虫肌幼虫培养不同时间后得到的ES
抗原组分大致相同，但也有不同的蛋白带。培养18、21、24、30小时得到的4种ES抗
原分子量相同的蛋白带为112、l一。、105、97、53、49、45、42、35、23、16kDa。4种
ES抗原中相同分子量蛋白的浓度也有差异，随着培养时间的延长，1 12、1 10、108 kDa
蛋白浓度逐渐降低，53、49、45 kDa蛋白浓度逐渐升高。在30hES抗原中存在而在18一24h
ES抗原中未发现的蛋白带为82、77、71、43、32kDa，其中82、32 kDa蛋白通过免疫
印迹证实不具有抗原性。在18h ES抗原中存在而在21一30 h ES抗原中未发现的蛋白带
为95kDa。
4.肌幼虫可溶性抗原与ES抗原的比较:旋毛虫肌幼虫ES抗原与可溶性抗原相同
蛋白带的分子量为112、110、108、102、97、53、49、45、42、35、23、16kDa，但ES
抗原较可溶性抗原蛋白带少。在肌幼虫ES抗原中存在而在可溶性抗原中未发现的蛋白
条带的分子量为82、77、71、32kDa。在可溶性抗原中存在而ES抗原中未发现的蛋白
条带为65、63、58、55、39、31、30、29、25、24、21、20、17、14、12 kDao
5.肌幼虫可溶性抗原与ES抗原中特异性组分的鉴定:在旋毛虫肌幼虫可溶性抗原
中只与旋毛虫感染的大鼠、小鼠及患者血清发生反应而不与其他寄生虫(并殖吸虫、华
支辜吸虫、日本血吸虫及囊虫)感染的动物或患者血清及正常人和小鼠血清发生反应的
蛋白条带为24、23、21、20 kDa，这些蛋白组分可作为血清学诊断旋毛虫病的特异性抗
原。18h ES抗原中的23 kDa和30h ES抗原中的71、23 kDa蛋白组分只与旋毛虫感染
的大鼠、小鼠及患者血清反应，而不与其它寄生虫感染的动物或患者血清及正常人和小
0鼠血清发生反应，故可作为诊断旋毛虫病的特异性抗原。
在肌幼虫18h ES抗原中102、97、95、53kDa蛋白和30h ES抗原中53、49、45kDa
蛋白可与其他寄生虫感染的动物或患者血清发生明显的交叉反应。因此，目前在欧美国
2
分
家用于旋毛虫病免疫学诊断的旋毛虫肌幼虫Es抗原的主要蛋白组分(45 kDa~sokDa及
43kDa)，在我国可能并不适用于旋毛虫病的免疫学诊断，因为肺吸虫、肝吸虫、日本血吸
虫和囊虫病在欧美国家少见，而这此寄生虫病在我国的感染率则较高，若将旋毛虫的上
述抗原组分用于旋毛虫病的免疫学诊断，则可与这些寄?
Trichinellosis is a serious parasitic zoonosis mainly caused by eating raw or undercooked pork and the meat of other animals containing Trichinella spiralis larvae. It is estimated that 11 million people in the world are infected by Trichinella. More than 10 000 cases of human trichinellosis were reported by the International Commission on Trichinellosis (ICT) from 1995 to 1997. Since human trichinellosis was found in China in 1964, 548 outbreaks of this disease has been occurred 12 Provinces/Autonomous Regions/Municipals (P/A/M) of China. Henan province is one of the high endemic areas of trichinellosis. Trichinellosis not only harms to human bodies but also causes huge economic loss. However, there are not satisfactory diagnostic techniques for this disease at present. The confirmed diagnosis relies on the muscle biopsy, but the larvae can not be detected in patients with light infection or in the earlier stage of infection, and the muscle biopsy is not easily accepted by patients. Hence, in order to resol
ve this problem, a lot of studies on immunodiagnosis of trichinellosis have been carried out at home and abroad. But, most of diagnostic antigens used in these studies were crude antigens of the encysted (muscle stage) larvae of T. spiralis. Because the antigenic components are very complicated, when the antigens were used for the immunodiagnosis of trichinellosis, they often occurred cross-reactions with sera from patients with other parasitic diseases (for example, paragonimiasis, clonorchiasis, schistosomiasis japonicum, cysticercosis and so on) and result in false-positive reactions. So it is very difficult to make a specific diagnosis for trichinellosis. In order to find specific antigens for the diagnosis of trichinellosis, the components of T. spiralis muscle larval soluble antigens and its excretory-secretory (ES) antigens were firstly analyzed by SDS-PAGE technology, then these antigens were used to react with sera from patients with trichinellosis, other other parasitosis and normal rats, mice and
persons by using immuno-blotting. In addition, in order to identify
the antigen causing cross reaction, adult worm soluble antigens of Paraonimus westermani and Clonorchis sinensis were analyzed too.
T. spiralis muscle larva, P. westermani adult and C. sinensis adult were homogenized, ultrasonicated, stirred, frozen and melted repeatedly, centrifugated, respectively, the supernate was collected and used as the worm soluble antigens. T. spiralis larvae were cultivated in serum-free RPMI-1640 medium for 18, 21, 24, 30 hours at 37C in CO2 incubator respectively. The culture fluids were collected, centrifugated, dialysed, lyophilized, respectively and T. spiralis muscle larva ES antigen were obtained. Protein contents of each antigens were assayed. These antigens were analyzed by the SDS-PAGE. 11% acrylamide separating gel and 4% acrylamide stacking gel were used in the electrophoresis. The gel was stained and bleached sufficiently, different electrophoresis bands were revealed. According to the migrating rate of standard molecule weight protein bands and the logarithm of molecule, standard curve was drawn, and then the corresponding molecule weight was checked out. Each antigens were analyzed by Western blot.
The results of SDS-PAGE and Western blot were described as follows.
1. Choice of protein contents of electrophoresis sample: there were differences among the electrophoresis bands, when the protein concentration was different in sample hole. The higher the sample concentration is, the less the quantity is needed, the better the bands is revealed, when electrophoresis system of Bio-Rad company was used, the protein content was about 14ug/hole; when electrophoresis system of Chinese company used, the protein content was about 25ug/hole. The amount of sample filled 1/4 of the hole, better results were acquired.
2. Analysis of T. spiralis muscle larva soluble antigen: muscle larva soluble antigen revealed the complicated band pattern consisted of 29 protein bands. The major bands with mole
将旋毛虫肌幼虫、卫氏并殖吸虫成虫和华支睾吸虫成虫分别研磨、超声破碎、冷浸、反复冻融、离心，制备虫体可溶性抗原；将旋毛虫肌幼虫在无血清的1640培养基中于5％的CO_2培养箱中分别培养18、21、24、30小时，分别离心、收集上清、透析，低温快速冰冻浓缩后得到培养18、21、24及30小时的旋毛虫肌幼虫ES抗原。测定各种抗原的蛋白含量。将上述各种抗原分别进行SDS-PAGE分析，电泳时用11％分离胶和4％积层胶。充分染色、脱色后可看到各自不同的电泳带。根据标准分子量蛋白带的迁移率及其分子量的对数绘制标准曲线，测量各条蛋白带迁移率，查出与其对应的分子量。然
后将各种抗原用Westem blot分析。SDS一PAGE电泳及Westem blot结果如下。
1.电泳样本蛋白含量的选择:加样孔内蛋白含量的不同，电泳带清晰度有所差异;
样品浓度越高，加样量越少，电泳结果越好;用Bi。一Rad公司的Mini电泳系统时，加
样量在14林岁孔左右;用国产的电泳系统时，加样量在25“g/孔左右。加样量在加样孔
高的1/4处时电泳效果最好。
2.肌幼虫可溶性抗原分析:旋毛虫肌幼虫可溶性抗原有29条蛋白带，主要分子
量范围在66切a~3 IkDa、20kDa~12切a之间，主要蛋白带为65、43、42、31、30、20、
17、16kDa。
3.培养不同时间的肌幼虫ES抗原分析:旋毛虫肌幼虫培养不同时间后得到的ES
抗原组分大致相同，但也有不同的蛋白带。培养18、21、24、30小时得到的4种ES抗
原分子量相同的蛋白带为112、l一。、105、97、53、49、45、42、35、23、16kDa。4种
ES抗原中相同分子量蛋白的浓度也有差异，随着培养时间的延长，1 12、1 10、108 kDa
蛋白浓度逐渐降低，53、49、45 kDa蛋白浓度逐渐升高。在30hES抗原中存在而在18一24h
ES抗原中未发现的蛋白带为82、77、71、43、32kDa，其中82、32 kDa蛋白通过免疫
印迹证实不具有抗原性。在18h ES抗原中存在而在21一30 h ES抗原中未发现的蛋白带
为95kDa。
4.肌幼虫可溶性抗原与ES抗原的比较:旋毛虫肌幼虫ES抗原与可溶性抗原相同
蛋白带的分子量为112、110、108、102、97、53、49、45、42、35、23、16kDa，但ES
抗原较可溶性抗原蛋白带少。在肌幼虫ES抗原中存在而在可溶性抗原中未发现的蛋白
条带的分子量为82、77、71、32kDa。在可溶性抗原中存在而ES抗原中未发现的蛋白
条带为65、63、58、55、39、31、30、29、25、24、21、20、17、14、12 kDao
5.肌幼虫可溶性抗原与ES抗原中特异性组分的鉴定:在旋毛虫肌幼虫可溶性抗原
中只与旋毛虫感染的大鼠、小鼠及患者血清发生反应而不与其他寄生虫(并殖吸虫、华
支辜吸虫、日本血吸虫及囊虫)感染的动物或患者血清及正常人和小鼠血清发生反应的
蛋白条带为24、23、21、20 kDa，这些蛋白组分可作为血清学诊断旋毛虫病的特异性抗
原。18h ES抗原中的23 kDa和30h ES抗原中的71、23 kDa蛋白组分只与旋毛虫感染
的大鼠、小鼠及患者血清反应，而不与其它寄生虫感染的动物或患者血清及正常人和小
0鼠血清发生反应，故可作为诊断旋毛虫病的特异性抗原。
在肌幼虫18h ES抗原中102、97、95、53kDa蛋白和30h ES抗原中53、49、45kDa
蛋白可与其他寄生虫感染的动物或患者血清发生明显的交叉反应。因此，目前在欧美国
2
分
家用于旋毛虫病免疫学诊断的旋毛虫肌幼虫Es抗原的主要蛋白组分(45 kDa~sokDa及
43kDa)，在我国可能并不适用于旋毛虫病的免疫学诊断，因为肺吸虫、肝吸虫、日本血吸
虫和囊虫病在欧美国家少见，而这此寄生虫病在我国的感染率则较高，若将旋毛虫的上
述抗原组分用于旋毛虫病的免疫学诊断，则可与这些寄?
Trichinellosis is a serious parasitic zoonosis mainly caused by eating raw or undercooked pork and the meat of other animals containing Trichinella spiralis larvae. It is estimated that 11 million people in the world are infected by Trichinella. More than 10 000 cases of human trichinellosis were reported by the International Commission on Trichinellosis (ICT) from 1995 to 1997. Since human trichinellosis was found in China in 1964, 548 outbreaks of this disease has been occurred 12 Provinces/Autonomous Regions/Municipals (P/A/M) of China. Henan province is one of the high endemic areas of trichinellosis. Trichinellosis not only harms to human bodies but also causes huge economic loss. However, there are not satisfactory diagnostic techniques for this disease at present. The confirmed diagnosis relies on the muscle biopsy, but the larvae can not be detected in patients with light infection or in the earlier stage of infection, and the muscle biopsy is not easily accepted by patients. Hence, in order to resol
ve this problem, a lot of studies on immunodiagnosis of trichinellosis have been carried out at home and abroad. But, most of diagnostic antigens used in these studies were crude antigens of the encysted (muscle stage) larvae of T. spiralis. Because the antigenic components are very complicated, when the antigens were used for the immunodiagnosis of trichinellosis, they often occurred cross-reactions with sera from patients with other parasitic diseases (for example, paragonimiasis, clonorchiasis, schistosomiasis japonicum, cysticercosis and so on) and result in false-positive reactions. So it is very difficult to make a specific diagnosis for trichinellosis. In order to find specific antigens for the diagnosis of trichinellosis, the components of T. spiralis muscle larval soluble antigens and its excretory-secretory (ES) antigens were firstly analyzed by SDS-PAGE technology, then these antigens were used to react with sera from patients with trichinellosis, other other parasitosis and normal rats, mice and
persons by using immuno-blotting. In addition, in order to identify
the antigen causing cross reaction, adult worm soluble antigens of Paraonimus westermani and Clonorchis sinensis were analyzed too.
T. spiralis muscle larva, P. westermani adult and C. sinensis adult were homogenized, ultrasonicated, stirred, frozen and melted repeatedly, centrifugated, respectively, the supernate was collected and used as the worm soluble antigens. T. spiralis larvae were cultivated in serum-free RPMI-1640 medium for 18, 21, 24, 30 hours at 37C in CO2 incubator respectively. The culture fluids were collected, centrifugated, dialysed, lyophilized, respectively and T. spiralis muscle larva ES antigen were obtained. Protein contents of each antigens were assayed. These antigens were analyzed by the SDS-PAGE. 11% acrylamide separating gel and 4% acrylamide stacking gel were used in the electrophoresis. The gel was stained and bleached sufficiently, different electrophoresis bands were revealed. According to the migrating rate of standard molecule weight protein bands and the logarithm of molecule, standard curve was drawn, and then the corresponding molecule weight was checked out. Each antigens were analyzed by Western blot.
The results of SDS-PAGE and Western blot were described as follows.
1. Choice of protein contents of electrophoresis sample: there were differences among the electrophoresis bands, when the protein concentration was different in sample hole. The higher the sample concentration is, the less the quantity is needed, the better the bands is revealed, when electrophoresis system of Bio-Rad company was used, the protein content was about 14ug/hole; when electrophoresis system of Chinese company used, the protein content was about 25ug/hole. The amount of sample filled 1/4 of the hole, better results were acquired.
2. Analysis of T. spiralis muscle larva soluble antigen: muscle larva soluble antigen revealed the complicated band pattern consisted of 29 protein bands. The major bands with mole
引文
1Dupouy-Camet J. Trichinellosis, a worldwide zoonosis[J]. Vet Parasitol, 2000, 93 (3-4) : 191-200．
2Murrell KD, Pozio E. Trichinellosis: the zoonosis that won't go quietly [J].Int J Parasitol, 2000,30(12-13) : 1339-1349．
3Wang ZQ,Cui J. Epidemiology of human trichinellosis in China during 1964-1999[J]. Parasite.2001,8 (2) : S63-S66．
4Wang ZQ, Cui J. Epidemiology of swine trichinellosis in China[J]. Parasite, 2001, 8(2) : S67-S70．
5Cui J,Wang ZQ, Wu F,et al. Epidemiological and clinical studies on an outbreak of trichinosis in Central China[J]. Ann Trop Med Parasitol,1997,91(5) :481-488．
6Wang ZQ,Cui J,Wu F, et al.Seven outbreaks of trichinosis in China during 1992-1996[J]. J Egypt Soc Parasitol, 1997,27(2) :529-538．
7Au AC, Ko RC, Simon JW, et al. Study of acute trichinosiis in Ghuskas: specificity and sensitivity of enzyme-linked immunosorbent assay for IgM and IgE antibodies to Trichinella larval antigens in diagnosis[J]. Trans R Soc Trop Med Hyg, 1983, 77(3) : 412-
415.
8 Chan SW, Ko RC. Comparison between standard ELISA and dot-ELISA for serodiagnosis of human trichinosis[J]. Trans R Soc Trop Med Hyg, 1988, 82(6): 892～894.
9 Niimura M, Kobayashi M, Kojima S. Immunodiagnositic value of an α-stichocyte derived antigen isolated by affinity chromatography for trichinosis[J]. Jap J Parasitol, 1992, 41(4): 287～291.
10 Maharmop P, Setasuban P, Morakote N, et al. Immunodiagnosis of human trichinellosis and identification of specific antigen for Trichinella spiralis[J]. Int J Parasitol, 1995, 25(1): 87～93.
11 Cui J, Wang ZQ, Wu F, et al. Fluorescent antibody technic for serodiagnosis of trichinosis [J]. Chin J Parasitol & Parasitic Dis, 1999, 16(1): 24～28.
12 曾庆仁，陆予云，易新元，等．日本血吸虫卵与旋毛虫肌蚴间相似抗原的研究[J]．中国寄生虫学与寄生虫病杂志，1997，15(6)：362～365．
13 曾庆仁，周金春，曾宪芳，等．旋毛虫肌虫幼可溶性抗原与日本血吸虫病患者血清的交叉反应[J]．湖南医科大学学报，1997，22(6)：475～477．
14 雷莉．冯瑞元．旋毛虫肌肉期幼虫膜抗原的制备及应用价值的初步探讨[J]．华西医大学报，1994，25(2)：5～8．
15 彭飞，曾宪芳，曾庆仁，等．旋毛虫脲溶性抗原与日本血吸虫抗体交叉反应的免疫印迹分析[J]．实用预防医学，1999，6(6)：426～427．
16 王中全，崔晶，晋雪香，等．琼脂凝胶双扩散试验诊断旋毛虫病的研究[J]．河南医学研究，1993，2(2)：138～140．
17 崔晶，武峰，王中全，等．胶乳凝集试验快速诊断旋毛虫病的研究[J]．中国卫生检验杂志，1995，3(5)：274～276
18 崔晶，王中全，晋雪香，等．ELISA对人体旋毛虫病诊断及疗效考核价值的研究[J]．河南医学研究，1996，5(1)：26～29．
19 崔晶，王中全，晋雪香，等．间接荧光抗体试验诊断人体旋毛虫病的研究[J]．中国寄生虫病防治杂志，1995，8(2)：137～139．
20 崔晶，王中全，晋雪香，等．河南省50例并殖吸虫病的流行病学和临床特征[J]．地方病通报，1997，12(2)：73～75．
21 王中全，崔晶，武峰，等．华支睾吸虫病14例临床分析[J]．胃肠病学与肝病学杂志，1997，
6(3)：278～279．
22 王中全，崔晶，晋雪香．旋毛虫幼虫收集方法的探讨[J]，河南医学研究，1993，2(1)：65～66．
23 陈兴旺，李调英，曾明安．斯氏虫3次提取的抗原单用及配用的诊断价值[J]．实用寄生虫病杂志，2000，8(3)：112～113．
24 王光西，许扬，毛樱逾，等．SDS-PAGE分析斯化肺吸虫幼虫和成虫排泄分泌抗原[J]．沪州医学院学报，2001，24(2)：103～104．
25 吴景兰，丁一主编．实用非放射性分子生物实验技术[M]．郑州：河南医科大学出版社，1997：142．
26 陈佩惠，周述龙主编．医学寄生虫体外培养[M]．北京：科学出版社，1995：52
27 Mahannop P, Chaicumpa W, Setasuban P, et al. Immunodiagnosis of human trichinellosis using excretory-secretory (ES) antigen[J]. J Helminthol, 1992, 66(4): 297～304.
28 周建新主编．现代医学生物实验基础教程[M]．北京：中国协和医科大学出版社，2000：32～34
29 金冬雁，黎孟枫，侯云德，等译．分子克隆实验指南[M]．第2版．北京：科学出版社，1992：880～898
30 朱正美，刘辉，主编．简明免疫学技术[M]．北京：科学出版社，2002：172～173．
31 李允鹤主编．寄生虫病免疫学及免疫诊断[M]．南京：江苏科学技术出版社，1991：417．
32 Herrera-Esparza R, del Rio-Castaneda A, Avalos-Diaz E, et al. Trichinella spiralis: immunochemical characterization of antigens in experimental infections[J]. Exp Parasitol, 1987, 63(2): 233～236.
33 Chan SW, Ko RC. Serodiagnosis of human trichinosis using a gel filtration antigen and indirect IgG-ELISA[J]. Trans R Soc Trop Med Hyg, 1990, 84(5): 721～722.
34 徐建余，程道新．旋毛虫感染期幼虫抗原的特异性分析[J]．中国寄生虫学与寄生虫病杂志，1998，16(2)：151～152．
35 Wang, ZQ, Cui J, Li HS, et al. Some observations on trichinosis in China[J]. Helminthologia, 1998, 35(1): 27～29.
36 彭飞，曾宪芳，曾庆仁，等．旋毛虫肌蚴可溶性抗原与日本血吸虫抗血清交叉反应的研究[J]．湖南医学，1999，16(3)：165～166
37 田明礼，易新元，曾宪芳，等．日本血吸虫不同发育阶段与旋毛虫抗原交叉性的研究[J]．湖南医科大学学报，1998，23(3)：225～228．
38Morakote N, Khamboonruang C, Siriprasert V, et al. The value of enzyme-linked immunosorbent assay (ELISA) for diagnosis of human trichinosis[J]. Trop Med Parasitol, 1991,42(3) :172-174．
39Dea-Ayuela MA, Bolas-Fernandez F. Trichinella antigens: a review[J]. Vet Res, 1999, 30(6) :559-571．
40Gamble HR, Anderson WR, Graham CE, et al. Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory-secretory antigen[J]. Vet Parasitol, 1983, 3(4) : 349-361．
41Su XZ, Prestwood AK. A dot-ELISA mimicry western blot test for the detection of swine trichinellosis[J]. J Parasitol,1991,77(1) :76-82．
42Zarlenga DS, Gamble HR. Molecular cloning and expression of an immunodominant 53-kDa excretory-secretory antigen from Trichinella spiralis muscle larvae[J]. Mol Biochem Parasitol, 1990,42(2) : 165-174．
43Gamble HR, Rapic D, Marinculic A, et al. Evaluation of excretory-secretory antigens for the serodiagnosis of swine trichinellosis[J]. Vet Parasitol,1988,30(2) :131-137．
44Murrell KD, Anderson WR, Schad GA, et al. Field evaluation of the enzyme-linked immunosorbent assay for swine trichinosis:efficacy of the excretory-secretory antigen[J]. Am J Vet Res, 1986, 47(5) : 1046-1049．
45Rapic D, Dzakula N, Matic-Piantanida D. Evaluation of different antigens in a seroepizootiological survey of trichinellosis by enzyme-linked immunosorbent assay[J]. Vet Parasitol,1986,21(4) :285-289．
46Zhu DZ, Bell RG. Trichinella spiralis: murine strain variation in response to monoclonally defined, protective, nonstage-specific antigens[J]. Exp Parasitol, 1990, 70 (3) :330-43
47Armas-Serra C, Gimenez-Pardo C, Jimenez-Gonzalez A, et al. Antibody response to a protease secreted by Trichinella spiralis muscle larvae[J]. Parasitol Res, 1995, 81(6) : 540-542．
48Moczon T, Wranicz M. Trichinella spiralis: proteinases in the larvae[J].Parasitol Res, 1999,85(1) : 47-58．
49Arden SR,Smith AM, Booth MJ,et al. Identification of serine/threonine protein kinases
secreted by Trichinella spiralis infective larvae[J]. Mol Biochem Parasitol, 1997, 90(1):111～119.
50 Todorova VK. Proteolytic enzymes secreted by larval stage of the parasitic nematode Trichinella spiralis[J]. Folia arasitol, 2000, 47(2): 141～145.
51 Rojas J, Rodriguez-Osorio M, Gomez-Garcia V. Immunological characteristics and localization of the Trichinella spiralis glutathione S-transferase[J]. J Parasitol, 1997, 83(4): 630～635.
52 Mak CH, Chung YY, Ko RC. Single-stranded endonuclease activity in the excretory-secretory products of Trichinella spiralis and Trichinella pseudospiralis[J]. Parasitol, 2000, 120(5): 527～533.
53 Bell RG. The generation and expression of immunity to Trichinella spiralis in laboratory rodents[J]. Adv Parasitol, 1998, 41: 150～217
54 朱兴全，窦兰清，史晓红，等．旋毛虫肌幼虫抗原可溶性抗原、排泄分泌抗原的电泳分析[J]．畜牧兽医学报，1995，26(2)：168～173．
55 李燎，雷莉，冯瑞元，等．旋毛虫肌肉期幼虫分泌排泄物中特异性抗原的分析[J]．地方病通报，1996，11(3)：15～18．
56 Gold AM, Despommier DD, Buck SW. Partial characterization of two antigens secreted by L1 larvae of Trichinella spiralis[J]. Mol Biochem Parasitol, 1990, 41(2): 187～196.
57 Leung RK, Ko RC. Production specific antigens from Trichinella spiralis using a continuous elution method and isoelectric focusing[J]. Southeast Asian J Trop Med Public Health, 1997, 28(Suppl 1): 99～106.
58 Nunez GG, Malmassari SL, Costantino SN, et al. Immunoelectrotransfer blot assay in acute and chronic human trichinellosis[J]. J Parasitol, 2000, 86(5): 1121～1124.
59 Wang ZQ, Cui J, Wu F, et al..Epidemiological study of paragonimiosis in central China[J]. Helminthologia, 1998, 35(3): 117～121.
60 王中全，马云祥，崔晶，等．河南省囊虫病的血清流行病学调查[J]．中国寄生虫病防治杂志，1999，12(3)：236～237
61 李调英，徐亮，曾明安．卫氏和斯氏肺吸虫抗原对同源及异源肺吸虫病诊断价值的比较[J]．实用寄生虫病杂志，2000，8(2)：86～87．
62 张锡林，李燎，陈文碧，等．斯氏肺吸虫成虫10～30kDa抗原蛋白的分离及在免疫诊断中
的应用[J]．泸州医学院学报，1996，19(3)：170～173．
63 Sugiyama H, Sugimoto M, Akasaka K, et al. Characterization and localization of Paragonimus westermani antigen stimulating antibody formation in both the infected cat and rat[J]. J Parasitol, 1987, 73(2): 363～367
64 Sugiyama H, Hinoue H, Katahira J, et al. Production of monoclonal antibody to characterize the antigen of Paragonimus westermani[J]. Parasitol Res, 1988, 75(2): 144～147
65 Slemenda SB, Maddison SE, Jong EC, et al. Diagnosis of paragonimiasis by immunoblot[J]. Am J Trop Med Hyg, 1988, 39(5): 469～471.
66 Kim SH, Kong Y, Kim SI, e al. Immunoblot observation of antigenic protein fractions in Paragonimus westermani reacting with huaman patients sera[J]. Korean J Parasitol, 1988, 26(4): 239～243.
67 张锡林，段建华,王英，等．斯氏狸殖吸虫抗原分析和血清学诊断方法的研究[J]．中国寄生虫学与寄生虫病杂志，2000，18(5)：277～281．
68 Wongkham C, Maleewong W, Intapan P, et al. Partially purified antigens of Paragonimus heterotremus for serodiagnosis of human paragonimiasis[J]. Southeast Asian J Trop Med Public Health, 1994, 25(1): 176～180.
69 Kong Y, Ito A, Yang HJ, et al. Immunoglobulin G (IgG) subclass and IgE responses in human paragonimiases caused by three different species[J]. Clin Diagn Lab Immunol, 1998, 5(4): 474～478.
70 Chung YB, Chung BS, Choi MH, et al. Partial characterization of a 17 kDa protein of Clonorchis sinensis. Korean J Parasitol, 2000, 38(2): 95～97.
71 张锡林，徐文岳，段建华，等．斯氏狸殖吸虫和华支睾吸虫基因组多态DNA的初步分析[J]．第三军医大学学报，2000，22(9)：865～867．
2Murrell KD, Pozio E. Trichinellosis: the zoonosis that won't go quietly [J].Int J Parasitol, 2000,30(12-13) : 1339-1349．
3Wang ZQ,Cui J. Epidemiology of human trichinellosis in China during 1964-1999[J]. Parasite.2001,8 (2) : S63-S66．
4Wang ZQ, Cui J. Epidemiology of swine trichinellosis in China[J]. Parasite, 2001, 8(2) : S67-S70．
5Cui J,Wang ZQ, Wu F,et al. Epidemiological and clinical studies on an outbreak of trichinosis in Central China[J]. Ann Trop Med Parasitol,1997,91(5) :481-488．
6Wang ZQ,Cui J,Wu F, et al.Seven outbreaks of trichinosis in China during 1992-1996[J]. J Egypt Soc Parasitol, 1997,27(2) :529-538．
7Au AC, Ko RC, Simon JW, et al. Study of acute trichinosiis in Ghuskas: specificity and sensitivity of enzyme-linked immunosorbent assay for IgM and IgE antibodies to Trichinella larval antigens in diagnosis[J]. Trans R Soc Trop Med Hyg, 1983, 77(3) : 412-
415.
8 Chan SW, Ko RC. Comparison between standard ELISA and dot-ELISA for serodiagnosis of human trichinosis[J]. Trans R Soc Trop Med Hyg, 1988, 82(6): 892～894.
9 Niimura M, Kobayashi M, Kojima S. Immunodiagnositic value of an α-stichocyte derived antigen isolated by affinity chromatography for trichinosis[J]. Jap J Parasitol, 1992, 41(4): 287～291.
10 Maharmop P, Setasuban P, Morakote N, et al. Immunodiagnosis of human trichinellosis and identification of specific antigen for Trichinella spiralis[J]. Int J Parasitol, 1995, 25(1): 87～93.
11 Cui J, Wang ZQ, Wu F, et al. Fluorescent antibody technic for serodiagnosis of trichinosis [J]. Chin J Parasitol & Parasitic Dis, 1999, 16(1): 24～28.
12 曾庆仁，陆予云，易新元，等．日本血吸虫卵与旋毛虫肌蚴间相似抗原的研究[J]．中国寄生虫学与寄生虫病杂志，1997，15(6)：362～365．
13 曾庆仁，周金春，曾宪芳，等．旋毛虫肌虫幼可溶性抗原与日本血吸虫病患者血清的交叉反应[J]．湖南医科大学学报，1997，22(6)：475～477．
14 雷莉．冯瑞元．旋毛虫肌肉期幼虫膜抗原的制备及应用价值的初步探讨[J]．华西医大学报，1994，25(2)：5～8．
15 彭飞，曾宪芳，曾庆仁，等．旋毛虫脲溶性抗原与日本血吸虫抗体交叉反应的免疫印迹分析[J]．实用预防医学，1999，6(6)：426～427．
16 王中全，崔晶，晋雪香，等．琼脂凝胶双扩散试验诊断旋毛虫病的研究[J]．河南医学研究，1993，2(2)：138～140．
17 崔晶，武峰，王中全，等．胶乳凝集试验快速诊断旋毛虫病的研究[J]．中国卫生检验杂志，1995，3(5)：274～276
18 崔晶，王中全，晋雪香，等．ELISA对人体旋毛虫病诊断及疗效考核价值的研究[J]．河南医学研究，1996，5(1)：26～29．
19 崔晶，王中全，晋雪香，等．间接荧光抗体试验诊断人体旋毛虫病的研究[J]．中国寄生虫病防治杂志，1995，8(2)：137～139．
20 崔晶，王中全，晋雪香，等．河南省50例并殖吸虫病的流行病学和临床特征[J]．地方病通报，1997，12(2)：73～75．
21 王中全，崔晶，武峰，等．华支睾吸虫病14例临床分析[J]．胃肠病学与肝病学杂志，1997，
6(3)：278～279．
22 王中全，崔晶，晋雪香．旋毛虫幼虫收集方法的探讨[J]，河南医学研究，1993，2(1)：65～66．
23 陈兴旺，李调英，曾明安．斯氏虫3次提取的抗原单用及配用的诊断价值[J]．实用寄生虫病杂志，2000，8(3)：112～113．
24 王光西，许扬，毛樱逾，等．SDS-PAGE分析斯化肺吸虫幼虫和成虫排泄分泌抗原[J]．沪州医学院学报，2001，24(2)：103～104．
25 吴景兰，丁一主编．实用非放射性分子生物实验技术[M]．郑州：河南医科大学出版社，1997：142．
26 陈佩惠，周述龙主编．医学寄生虫体外培养[M]．北京：科学出版社，1995：52
27 Mahannop P, Chaicumpa W, Setasuban P, et al. Immunodiagnosis of human trichinellosis using excretory-secretory (ES) antigen[J]. J Helminthol, 1992, 66(4): 297～304.
28 周建新主编．现代医学生物实验基础教程[M]．北京：中国协和医科大学出版社，2000：32～34
29 金冬雁，黎孟枫，侯云德，等译．分子克隆实验指南[M]．第2版．北京：科学出版社，1992：880～898
30 朱正美，刘辉，主编．简明免疫学技术[M]．北京：科学出版社，2002：172～173．
31 李允鹤主编．寄生虫病免疫学及免疫诊断[M]．南京：江苏科学技术出版社，1991：417．
32 Herrera-Esparza R, del Rio-Castaneda A, Avalos-Diaz E, et al. Trichinella spiralis: immunochemical characterization of antigens in experimental infections[J]. Exp Parasitol, 1987, 63(2): 233～236.
33 Chan SW, Ko RC. Serodiagnosis of human trichinosis using a gel filtration antigen and indirect IgG-ELISA[J]. Trans R Soc Trop Med Hyg, 1990, 84(5): 721～722.
34 徐建余，程道新．旋毛虫感染期幼虫抗原的特异性分析[J]．中国寄生虫学与寄生虫病杂志，1998，16(2)：151～152．
35 Wang, ZQ, Cui J, Li HS, et al. Some observations on trichinosis in China[J]. Helminthologia, 1998, 35(1): 27～29.
36 彭飞，曾宪芳，曾庆仁，等．旋毛虫肌蚴可溶性抗原与日本血吸虫抗血清交叉反应的研究[J]．湖南医学，1999，16(3)：165～166
37 田明礼，易新元，曾宪芳，等．日本血吸虫不同发育阶段与旋毛虫抗原交叉性的研究[J]．湖南医科大学学报，1998，23(3)：225～228．
38Morakote N, Khamboonruang C, Siriprasert V, et al. The value of enzyme-linked immunosorbent assay (ELISA) for diagnosis of human trichinosis[J]. Trop Med Parasitol, 1991,42(3) :172-174．
39Dea-Ayuela MA, Bolas-Fernandez F. Trichinella antigens: a review[J]. Vet Res, 1999, 30(6) :559-571．
40Gamble HR, Anderson WR, Graham CE, et al. Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory-secretory antigen[J]. Vet Parasitol, 1983, 3(4) : 349-361．
41Su XZ, Prestwood AK. A dot-ELISA mimicry western blot test for the detection of swine trichinellosis[J]. J Parasitol,1991,77(1) :76-82．
42Zarlenga DS, Gamble HR. Molecular cloning and expression of an immunodominant 53-kDa excretory-secretory antigen from Trichinella spiralis muscle larvae[J]. Mol Biochem Parasitol, 1990,42(2) : 165-174．
43Gamble HR, Rapic D, Marinculic A, et al. Evaluation of excretory-secretory antigens for the serodiagnosis of swine trichinellosis[J]. Vet Parasitol,1988,30(2) :131-137．
44Murrell KD, Anderson WR, Schad GA, et al. Field evaluation of the enzyme-linked immunosorbent assay for swine trichinosis:efficacy of the excretory-secretory antigen[J]. Am J Vet Res, 1986, 47(5) : 1046-1049．
45Rapic D, Dzakula N, Matic-Piantanida D. Evaluation of different antigens in a seroepizootiological survey of trichinellosis by enzyme-linked immunosorbent assay[J]. Vet Parasitol,1986,21(4) :285-289．
46Zhu DZ, Bell RG. Trichinella spiralis: murine strain variation in response to monoclonally defined, protective, nonstage-specific antigens[J]. Exp Parasitol, 1990, 70 (3) :330-43
47Armas-Serra C, Gimenez-Pardo C, Jimenez-Gonzalez A, et al. Antibody response to a protease secreted by Trichinella spiralis muscle larvae[J]. Parasitol Res, 1995, 81(6) : 540-542．
48Moczon T, Wranicz M. Trichinella spiralis: proteinases in the larvae[J].Parasitol Res, 1999,85(1) : 47-58．
49Arden SR,Smith AM, Booth MJ,et al. Identification of serine/threonine protein kinases
secreted by Trichinella spiralis infective larvae[J]. Mol Biochem Parasitol, 1997, 90(1):111～119.
50 Todorova VK. Proteolytic enzymes secreted by larval stage of the parasitic nematode Trichinella spiralis[J]. Folia arasitol, 2000, 47(2): 141～145.
51 Rojas J, Rodriguez-Osorio M, Gomez-Garcia V. Immunological characteristics and localization of the Trichinella spiralis glutathione S-transferase[J]. J Parasitol, 1997, 83(4): 630～635.
52 Mak CH, Chung YY, Ko RC. Single-stranded endonuclease activity in the excretory-secretory products of Trichinella spiralis and Trichinella pseudospiralis[J]. Parasitol, 2000, 120(5): 527～533.
53 Bell RG. The generation and expression of immunity to Trichinella spiralis in laboratory rodents[J]. Adv Parasitol, 1998, 41: 150～217
54 朱兴全，窦兰清，史晓红，等．旋毛虫肌幼虫抗原可溶性抗原、排泄分泌抗原的电泳分析[J]．畜牧兽医学报，1995，26(2)：168～173．
55 李燎，雷莉，冯瑞元，等．旋毛虫肌肉期幼虫分泌排泄物中特异性抗原的分析[J]．地方病通报，1996，11(3)：15～18．
56 Gold AM, Despommier DD, Buck SW. Partial characterization of two antigens secreted by L1 larvae of Trichinella spiralis[J]. Mol Biochem Parasitol, 1990, 41(2): 187～196.
57 Leung RK, Ko RC. Production specific antigens from Trichinella spiralis using a continuous elution method and isoelectric focusing[J]. Southeast Asian J Trop Med Public Health, 1997, 28(Suppl 1): 99～106.
58 Nunez GG, Malmassari SL, Costantino SN, et al. Immunoelectrotransfer blot assay in acute and chronic human trichinellosis[J]. J Parasitol, 2000, 86(5): 1121～1124.
59 Wang ZQ, Cui J, Wu F, et al..Epidemiological study of paragonimiosis in central China[J]. Helminthologia, 1998, 35(3): 117～121.
60 王中全，马云祥，崔晶，等．河南省囊虫病的血清流行病学调查[J]．中国寄生虫病防治杂志，1999，12(3)：236～237
61 李调英，徐亮，曾明安．卫氏和斯氏肺吸虫抗原对同源及异源肺吸虫病诊断价值的比较[J]．实用寄生虫病杂志，2000，8(2)：86～87．
62 张锡林，李燎，陈文碧，等．斯氏肺吸虫成虫10～30kDa抗原蛋白的分离及在免疫诊断中
的应用[J]．泸州医学院学报，1996，19(3)：170～173．
63 Sugiyama H, Sugimoto M, Akasaka K, et al. Characterization and localization of Paragonimus westermani antigen stimulating antibody formation in both the infected cat and rat[J]. J Parasitol, 1987, 73(2): 363～367
64 Sugiyama H, Hinoue H, Katahira J, et al. Production of monoclonal antibody to characterize the antigen of Paragonimus westermani[J]. Parasitol Res, 1988, 75(2): 144～147
65 Slemenda SB, Maddison SE, Jong EC, et al. Diagnosis of paragonimiasis by immunoblot[J]. Am J Trop Med Hyg, 1988, 39(5): 469～471.
66 Kim SH, Kong Y, Kim SI, e al. Immunoblot observation of antigenic protein fractions in Paragonimus westermani reacting with huaman patients sera[J]. Korean J Parasitol, 1988, 26(4): 239～243.
67 张锡林，段建华,王英，等．斯氏狸殖吸虫抗原分析和血清学诊断方法的研究[J]．中国寄生虫学与寄生虫病杂志，2000，18(5)：277～281．
68 Wongkham C, Maleewong W, Intapan P, et al. Partially purified antigens of Paragonimus heterotremus for serodiagnosis of human paragonimiasis[J]. Southeast Asian J Trop Med Public Health, 1994, 25(1): 176～180.
69 Kong Y, Ito A, Yang HJ, et al. Immunoglobulin G (IgG) subclass and IgE responses in human paragonimiases caused by three different species[J]. Clin Diagn Lab Immunol, 1998, 5(4): 474～478.
70 Chung YB, Chung BS, Choi MH, et al. Partial characterization of a 17 kDa protein of Clonorchis sinensis. Korean J Parasitol, 2000, 38(2): 95～97.
71 张锡林，徐文岳，段建华，等．斯氏狸殖吸虫和华支睾吸虫基因组多态DNA的初步分析[J]．第三军医大学学报，2000，22(9)：865～867．