【作者】 张灯；

【导师】 王中全； 崔晶；

【作者基本信息】 郑州大学 ， 病原生物学， 2003， 硕士

【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CO₂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a³ IkDa、20kDa¹2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18一24hES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?更多还原

【Abstract】 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 resolve 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 identifythe 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更多还原