羊泰勒虫裂殖子蛋白分析及其ELISA诊断方法的建立

【作者】 高玉龙；

【导师】 欧阳五庆； 殷宏； 罗建勋；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2001， 硕士

【摘要】 羊泰勒虫不同地理分布的虫株在致病力上有很大差异，但不同虫株在蛋白多肽及抗原特性方面是否存在差异，尚不得而知，为此本试验利用十二烷基硫酸钠—聚丙烯酰胺凝胶电泳（SDS—PAGE）和免疫印迹（Western blotting）方法对羊泰勒虫甘南株（甘肃省）、隆德株（宁夏回族自治区）、张家川株（甘肃省）和麻当株（甘肃省）虫体蛋白进行了分析。SDS—PAGE结果显示，羊泰勒虫甘南株出现了16条带，粗带有102、71、41、33、29、 14kD；隆德株出现了12条带，粗带有102、71、41、31、29、14kD；张家川株出现了14条带，粗带有102、38、33、22、17、16、14kD；麻当株出现了12条带，粗带有102、38、37、27、17、16、14kD。4个虫株蛋白多肽在数量、含量和分子量上存在差异。对4个虫株的电泳谱进行了比较，发现羊泰勒虫甘南株与隆德株的亲源关系较近，与张家川株的亲源关系次之；张家川株与麻当株的亲源关系也比较近。4个虫株的免疫血清分别与4株虫体蛋白进行了免疫印迹试验，结果表明4株虫体的免疫血清与甘南株和张家川株在38kD处有共同反应。免疫印迹试验还发现了隆德株虫体在38kD的多肽和麻当株虫体在35kD的多肽。 自感染红细胞内纯化的羊泰勒虫裂殖子，经冻融、超声裂解后，其可溶性抗原过SephadexG-200，收集蛋白峰，以此作为包被抗原，用常规方法建立了羊泰勒虫病的间接ELISA诊断方法。特异性试验和敏感性试验证明此法特异性高、重复性好。对46份阳性血清和48份阴性血清用ELISA检测，结果阳性检出率为100％，阴性符合率为98.0％。用此方法对人工感染羊和野外感染羊的抗体消长进行检测，结果显示，人工感染羊于感染后第5d即可检出抗体，30～40d达到高峰，抗体液度为1∶3200，然后下降，并维持在1∶400～1∶800之间；野外自然感染羊的抗体水平出现了2个峰值，第1峰在3～7月份之间，第2峰在9～10月份之间。对甘肃省甘南藏族自治州玛曲县、甘南藏族自治州临洮县、张家川回族自治县和天祝藏族自治县的血清进行了检测，其阳性率分别为5.7％、93.8％、82.5％和80.0％，与现可查流行病学调查基本一致。更多还原

【Abstract】 There was clear difference in pathogenic ability of Theileria sp. isolated from sheep in different districts in China. Merozoite proteins of four strains of Thelleria sp. (Gannan strain, Longde strain, Zhangjiachuan strain and Madang strain) were analyzed with SDS ?PAGE and Western blotting in order to ascertain whether there are differences in protein polypeptides and characteristics of antigen of Theileria sp. isolated from sheep in different districts. SDS ?PAGE indicated that 16 bands were shown in the lane of Thelleria sp. (Gannan strain), including 6 wide bands: 102, 71, 41, 33, 29 and l4kD; 12 bands of Theileria sp. (Longde strain), including 6 wide bands: 102, 71, 41, 31, 29 and I4kD; 14 bands of Theileria sp. (Zhangjiachuan strain), including 7 wide bands: 102, 38, 33, 22, 17, 16 and I4kD; 12 bands of Theileria sp. (Madang strain), including 7 wide bands: 102, 38, 37, 27, 17, 16 and l4kD. There were differences in the number, content and molecular weight of protein polypeptides. The lanes of electrophoresis of four strains of The ileria sp. were analyzed. The results indicated that Gannan strain and Longde strain of Theileria sp. had a very close relationship, that Gannan strain and Zhangjiachuan strain had a close relationship, and that Zhangjiachuan strain and Madang strain had a very close relationship. When the proteins were transferred to PVDF membrane and probed with positive sheep sera against those four strains of Theileria sp、The positive sheep sera against those four strains of Theileria sp. reacted all with the protein polypeptides of Gannan strain and Zhangjiachuan strain in 38kD. The protein polypeptides of Longde strain in 38kD and Madang strain in 35kD were found in the experiment of Western blotting. An indirect enzyme-linked imrnunosorbent assay (ELISA) was first reported for successfully detecting specific antibody to Theileria sp.. The merozoite antigen was used to coat ELISA plates. The optimal condition (concentration of antigen, serum and conjugate) of the ELISA was evaluated. Furthermore, the coating time, Tween-20 concentration, dilution and wash were evaluated. The cut-off value was determined by the mean of negative control plus three standard deviation. The positive coincident rate of the ELISA was 100% according to the results examining 46 positive sera and the negative coincident rate of the ELISA was 98.0% according to the results examining 48 negative sera. The antibody level of sheep experimentally and naturally infected with Theileria sp. was examined by the ELISA. The antibody to Thelleria sp. could be detected by the ELISA in two sheep experimentally infected at 5 days post-infection. The peak antibody levels (1:3200) reacted between 30 and 40 days post-infection and then gradually fell to I :400-1:800. The antibody could be detected at 270 days post- infection. The antibody levels reached two peaks in two naturally infected sheep. The first peak was reached between March and July and the other was reached between September and October. The antibody to Theileria sp. was detected in samples obtained from four counties of Gansu province (including Madang County, Lintaxi County, Zhangjiachuan County and Tianzhu County) by the ELISA. The positive-rates was respectively 5.7%, 93.80/0, 82.5% and 80.0%.更多还原