多房棘球绦虫可溶性抗原分离纯化及免疫学鉴定

发布时间：2018-06-11 16:12

  本文选题：多房棘球绦虫 + 分离纯化　； 参考：《青海大学》2017年硕士论文

【摘要】：目的主要对多房棘球绦虫原头节可溶性蛋白进行分离纯化并富集蛋白微量组份,并对各组份进行免疫学鉴定,获得可用于多房棘球蚴病早期诊断的特异性抗原,进一步对其特异性抗原的二级结构及优势抗原表位进行鉴定、分析和预测。方法1.通过使用HiTrap Q FF及Mono Q 5/50 GL阴离子交换柱,将多房棘球绦虫原头节可溶性抗原进行分离,再将各组份抗原与多房棘球蚴患者、细粒棘球蚴患者及健康人血清进行免疫印迹及质谱鉴定,进一步通过BLAST等生物信息学方法比对以获得特异性高的多房棘球蚴抗原。2.将获得的特异性抗原氨基酸序列通过生物信息学软件SOPMA预测其蛋白二级结构,进一步通过生物信息学软件IEDB、SYFPEITHI、Bcepred和ABCpred预测其潜在的T细胞和B细胞表位。结果1.经HiTrap Q FF及Mono Q 5/50 GL阴离子交换柱富集共获得9个组份,进一步Western-blot结果显示在55kDa处具有良好免疫原性及较高特异性的目的蛋白,经质谱鉴定后,根据其物种来源、得分、氨基酸覆盖率、总氨基酸数、蛋白分子量等筛选出20个蛋白(Top20),进一步对Top20蛋白序列进行BLAST比对,发现多房棘球绦虫来源亮氨酰氨肽酶(leucyl aminopeptidase,LAP)与细粒棘球绦虫来源的氨基酸序列差异最大,相似度为62.63%,且无相似序列的人源及其他寄生虫来源蛋白。2.分析出多房棘球绦虫亮氨酰氨肽酶的蛋白质二级结构中α螺旋占比为45.42%、β折叠占比为16.57%、β转角占比为8.58%、无规则卷曲占比为29.43%。预测出亮氨酰氨肽酶具有9个T细胞潜在优势抗原表位,分别为L65-73、L75-84、L174-186、L233-240、L301-308、L333-343、L392-400、L445-454、L482-490;预测出多房棘球绦虫亮氨酰氨肽酶具有20个B细胞潜在优势抗原表位,分别为L13-22、L39-47、L75-84、L98-110、L146-154、L174-186、L183-191、L233-240、L247-254、L281-288、L295-306、L319-328、L330-337、L339-347、L392-400、L397-410、L421-428、L464-473、L493-504、L504-512。结论1.多房棘球绦虫中LAP具有较高的特异性,其氨基酸序列与细粒棘球绦虫及其他寄生虫相似度较低,可能是潜在的多房棘球蚴病免疫诊断的理想候选抗原;2.多房棘球绦虫亮氨酰氨肽酶具有9个T细胞潜在优势抗原表位及20个B细胞潜在优势抗原表位,并且其中存在4个同时具有B/T双细胞表位,分别为L75-84、L174-186、L233-240、L392-400。可为后续多房棘球绦虫相关亮氨酰氨肽酶的血清学检测、免疫预防及多表位疫苗研发奠定理论基础。
[Abstract]:Objective to isolate and purify the soluble protein of Echinococcus multilocularis and enrich the trace components of the protein, and to obtain the specific antigen which can be used for early diagnosis of Echinococcus multilocularis. Furthermore, the secondary structure and epitopes of its specific antigen were identified, analyzed and predicted. Method 1. By using HiTrap Q FF and Mono Q 5 / 50 GL anion exchange column, the soluble antigen of Echinococcus multilocularis in the head ganglia was isolated. The sera of patients with Echinococcus granulosus and healthy persons were identified by Western blot and mass spectrometry, and the specific multilocular-echinococcal antigen .2was obtained by comparison with other bioinformatics methods such as blast. The protein secondary structure was predicted by bioinformatics software SOPMA, and its potential T and B cell epitopes were predicted by bioinformatics software IEDB-SYFPEITHIBcepred and ABCpred. Result 1. Nine components were obtained by HiTrap Q FF and Mono Q 5 / 50 GL anion exchange column. Further Western-blot analysis showed that the target protein had good immunogenicity and high specificity at 55kDa. Amino acid coverage, total amino acid number, molecular weight of protein, etc., were screened out 20 proteins named Top20, and the sequence of Top20 protein was further compared with blast. It was found that the amino acid sequence of leucyl aminopeptidase (LAPs) from Echinococcus multilocularis and Echinococcus granulosus was the most different, and the similarity was 62.63 and the human and other parasitic proteins. The protein secondary structure of Leucoylaminopeptidase of Echinococcus multilocularis was analyzed. The percentage of 伪 helix was 45.42, the percentage of 尾 folding was 16.57, the proportion of 尾 turn angle was 8.58 and the proportion of irregular crimp was 29.43. 棰勬祴鍑轰寒姘ㄩ叞姘ㄨ偨閰跺叿鏈，

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