本文選題：多房棘球絳蟲 + 分離純化　； 參考：《青海大學(xué)》2017年碩士論文

【摘要】：目的主要對(duì)多房棘球絳蟲原頭節(jié)可溶性蛋白進(jìn)行分離純化并富集蛋白微量組份,并對(duì)各組份進(jìn)行免疫學(xué)鑒定,獲得可用于多房棘球蚴病早期診斷的特異性抗原,進(jìn)一步對(duì)其特異性抗原的二級(jí)結(jié)構(gòu)及優(yōu)勢(shì)抗原表位進(jìn)行鑒定、分析和預(yù)測(cè)。方法1.通過(guò)使用HiTrap Q FF及Mono Q 5/50 GL陰離子交換柱,將多房棘球絳蟲原頭節(jié)可溶性抗原進(jìn)行分離,再將各組份抗原與多房棘球蚴患者、細(xì)粒棘球蚴患者及健康人血清進(jìn)行免疫印跡及質(zhì)譜鑒定,進(jìn)一步通過(guò)BLAST等生物信息學(xué)方法比對(duì)以獲得特異性高的多房棘球蚴抗原。2.將獲得的特異性抗原氨基酸序列通過(guò)生物信息學(xué)軟件SOPMA預(yù)測(cè)其蛋白二級(jí)結(jié)構(gòu),進(jìn)一步通過(guò)生物信息學(xué)軟件IEDB、SYFPEITHI、Bcepred和ABCpred預(yù)測(cè)其潛在的T細(xì)胞和B細(xì)胞表位。結(jié)果1.經(jīng)HiTrap Q FF及Mono Q 5/50 GL陰離子交換柱富集共獲得9個(gè)組份,進(jìn)一步Western-blot結(jié)果顯示在55kDa處具有良好免疫原性及較高特異性的目的蛋白,經(jīng)質(zhì)譜鑒定后,根據(jù)其物種來(lái)源、得分、氨基酸覆蓋率、總氨基酸數(shù)、蛋白分子量等篩選出20個(gè)蛋白(Top20),進(jìn)一步對(duì)Top20蛋白序列進(jìn)行BLAST比對(duì),發(fā)現(xiàn)多房棘球絳蟲來(lái)源亮氨酰氨肽酶(leucyl aminopeptidase,LAP)與細(xì)粒棘球絳蟲來(lái)源的氨基酸序列差異最大,相似度為62.63%,且無(wú)相似序列的人源及其他寄生蟲來(lái)源蛋白。2.分析出多房棘球絳蟲亮氨酰氨肽酶的蛋白質(zhì)二級(jí)結(jié)構(gòu)中α螺旋占比為45.42%、β折疊占比為16.57%、β轉(zhuǎn)角占比為8.58%、無(wú)規(guī)則卷曲占比為29.43%。預(yù)測(cè)出亮氨酰氨肽酶具有9個(gè)T細(xì)胞潛在優(yōu)勢(shì)抗原表位,分別為L(zhǎng)65-73、L75-84、L174-186、L233-240、L301-308、L333-343、L392-400、L445-454、L482-490;預(yù)測(cè)出多房棘球絳蟲亮氨酰氨肽酶具有20個(gè)B細(xì)胞潛在優(yōu)勢(shì)抗原表位,分別為L(zhǎng)13-22、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。結(jié)論1.多房棘球絳蟲中LAP具有較高的特異性,其氨基酸序列與細(xì)粒棘球絳蟲及其他寄生蟲相似度較低,可能是潛在的多房棘球蚴病免疫診斷的理想候選抗原;2.多房棘球絳蟲亮氨酰氨肽酶具有9個(gè)T細(xì)胞潛在優(yōu)勢(shì)抗原表位及20個(gè)B細(xì)胞潛在優(yōu)勢(shì)抗原表位,并且其中存在4個(gè)同時(shí)具有B/T雙細(xì)胞表位,分別為L(zhǎng)75-84、L174-186、L233-240、L392-400�？蔀楹罄m(xù)多房棘球絳蟲相關(guān)亮氨酰氨肽酶的血清學(xué)檢測(cè)、免疫預(yù)防及多表位疫苗研發(fā)奠定理論基礎(chǔ)。
[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 偽 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. 棰勬祴鍑轟寒姘ㄩ叞姘ㄨ偨閰跺叿鏈，

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