本文關(guān)鍵詞：日本血吸蟲體被蛋白作為診斷和疫苗候選抗原的研究　出處：《中國疾病預防控制中心》2009年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 日本血吸蟲 體被 Sjannexin Sj22 Sjinnexin SjMRLC 診斷 疫苗

【摘要】： 血吸蟲病仍然是影響人類健康的重要寄生蟲病�，F(xiàn)有的免疫學診斷方法主要是用血吸蟲成蟲粗抗原或蟲卵粗抗原檢測抗體,材料來源受限,且不能區(qū)分現(xiàn)癥感染和既往感染,特異性也有待進一步提高。血吸蟲疫苗一直是研究熱點,但目前仍無突破性進展。血吸蟲體被是血吸蟲和宿主相互作用的兩個主要界面之一,在營養(yǎng)吸收和免疫逃避等多個方面發(fā)揮重要作用,被認為是診斷、疫苗和藥物的潛在靶點。前期日本血吸蟲的體被蛋白質(zhì)組學研究鑒定了一批體被蛋白質(zhì)。本研究就是針對其中的Sjannexin(AY813612,AAW25344)、Sj22(AY815492,AAW27224)、Sjinnexin(AY815599,AAW27331)、SjMRLC(AY815219,AAW26951)等4個蛋白質(zhì)開展相關(guān)研究,主要研究內(nèi)容包括:(1)利用已知的序列進行生物信息學分析,如:信號肽預測、跨膜區(qū)域預測、功能位點和結(jié)構(gòu)域預測、同源序列搜索、多序列比對和進化樹構(gòu)建等;(2)基因的原核克隆和表達、重組蛋白質(zhì)的純化;(3)重組蛋白免疫原性檢測,重組蛋白兔多克隆抗體的制備及評價,基因的蟲期轉(zhuǎn)錄特異性,蛋白質(zhì)的蟲期表達特異性,蛋白質(zhì)的免疫定位;(4)蛋白質(zhì)的診斷價值評價和蛋白質(zhì)的動物保護性實驗。 Sjannexin是相對保守的蛋白,與日本血吸蟲重要宿主中的同源序列相似性較高,含有4個annexin重復單位結(jié)構(gòu)域;構(gòu)建了pET-28a/Sjannexin重組質(zhì)粒,誘導表達獲得可溶性蛋白,通過含咪唑的洗脫液純化,獲得了較高濃度和純度的重組蛋白;Western blot檢測表明rSjannexin具有較好的免疫原性;rSjannexin免疫兔多克隆抗體的滴度達到1:256,000;Sjannexin基因在童蟲、雌蟲、雄蟲和合抱成蟲中轉(zhuǎn)錄水平較高,胞蚴、毛蚴、蟲卵中轉(zhuǎn)錄水平較低,尾蚴中轉(zhuǎn)錄水平最低;Sjannexin主要在尾蚴、童蟲、雌蟲、雄蟲和合抱成蟲中表達,而在蟲卵中不表達;Sjannexin定位于日本血吸蟲雄蟲的體被;rSjannexin作為包被抗原,采用生物素親和素放大系統(tǒng)(BAS-ELISA)檢測不同人群血清,敏感度和特異度分別為80%和90%;用rSjannexin免疫小鼠獲得了3.82%的減蟲率,肝臟減卵率為21.13%,感染后40d～42d糞便減卵率為31.44%,感染后43d～45d糞便減卵率為24.17%,但統(tǒng)計學檢驗均無差異(p＞0.05)。 Sj22是日本血吸蟲的特有蛋白,含有1個信號肽;構(gòu)建了pET-28a/Sj22重組質(zhì)粒,誘導表達獲得不可溶性蛋白,通過尿素純化,獲得了較高濃度和純度的重組蛋白;Western blot檢測顯示rSj22具有一定的免疫原性;rSj22免疫兔多克隆抗體的滴度達到1:256,000;Sj22基因在雌蟲、雄蟲和合抱成蟲中轉(zhuǎn)錄水平較高,胞蚴、毛蚴、尾蚴和童蟲中轉(zhuǎn)錄水平較低,而蟲卵中無轉(zhuǎn)錄;Sj22主要在雄蟲和合抱成蟲中表達,在雌蟲期有少量表達,提示Sj22可能是性別相關(guān)基因;免疫定位顯示Sj22表達于日本血吸蟲雄蟲的腸腔,可能是分泌蛋白,這與Sj22含有信號肽相一致;rSj22作為包被抗原,采用生物素親和素放大系統(tǒng)(BAS-ELISA)檢測不同人群血清,敏感度和特異度分別為36.7%和80%;用rSj22免疫小鼠獲得了17.28%的減蟲率和9.03%的肝臟減卵率,感染后43d～45d糞便減卵率為22.53%,但統(tǒng)計學檢驗均無差異(p＞0.05)。 Sjinnexin與日本血吸蟲重要宿主中的同源序列的相似性較低;本研究在原核表達系統(tǒng)中克隆并表達了Sjinnexin的第二個胞外環(huán)(loop2,AA228～AA331),獲得不可溶性蛋白,通過尿素純化,獲得了較高濃度和純度重組抗原片段;Western blot檢測顯示日本血吸蟲感染動物血清(鼠和兔)及日本血吸蟲患者血清均較正常動物血清和人血清有不同程度增強,表明其具有較好的免疫原性;Sjinnexin基因在胞蚴、毛蚴、蟲卵、尾蚴、童蟲、雌蟲、雄蟲和合抱成蟲中均有轉(zhuǎn)錄,且水平比較穩(wěn)定;用rSjinnexin免疫小鼠獲得了12.21%的減蟲率(p＞0.05),肝臟減卵率為32.98%(p＞0.05),感染后40d～42d糞便減卵率為61.32%(p＜0.05),感染后43d～45d糞便減卵率為53.55%(p＞0.05),說明其具有一定的抗生殖作用,是潛在的抗生殖疫苗候選靶標。 SjMRLC同日本血吸蟲重要宿主中的同源序列在進化上屬于不同的分支,SjMRLC含有1個EF-hand Ca~(2+)結(jié)合結(jié)構(gòu)域,提示其具有Ca~(2+)結(jié)合功能;本研究構(gòu)建了pGEX-4T-3/SjMRLC重組質(zhì)粒,獲得可溶性蛋白;SjMRLC基因在胞蚴、毛蚴、蟲卵、尾蚴、童蟲、雌蟲、雄蟲和合抱成蟲中均有轉(zhuǎn)錄,且水平比較穩(wěn)定。
[Abstract]:Schistosomiasis is still an important parasitic disease to human health. The immunological diagnostic method is mainly used in adult Schistosoma eggs or crude antigen crude antigen antibody detection, the material source is limited, and can not distinguish between current infection and previous infection, specific also needs to be further improved. Schistosomiasis vaccine has been a hot research topic, but there is still no breakthrough. Schistosoma body is one of the two main interface of Schistosoma and host interactions, in the absorption of nutrients and immune escape play an important role in many aspects, is considered to be a potential target for diagnosis, vaccine and medicine. Early Schistosoma japonicum tegumental proteomics research identified a number of proteins. The body is study on the Sjannexin (AY813612, AAW25344), Sj22 (AY815492, AAW27224), Sjinnexin (AY815599, AAW27331), SjMRLC (AY815219, AAW26951) 4 protein Carry out the related research, the main research contents include: (1) using the known sequence by bioinformatics analysis, such as: signal peptide prediction, the prediction of transmembrane region, functional sites and domain prediction, homology search, multiple sequence alignment and phylogenetic tree construction; (2) gene and prokaryotic expression clone the purified recombinant protein, the recombinant protein; (3) to detect the immunogenicity of recombinant protein, rabbit polyclonal antibody preparation and evaluation, stage specific gene transcription, specific protein expression stage, immunohistochemical localization of the protein; (4) to evaluate the diagnostic value of protein and protein of animal protection experiment.
Sjannexin is a relatively conservative protein, high similarity with homologous sequences of Schistosoma japonicum important host, containing 4 annexin repeat domain; recombinant plasmid pET-28a/Sjannexin was constructed, which induced expression of soluble protein, the purified eluent containing imidazole, the recombinant protein with high concentration and purity; Western blot assay showed that rSjannexin it has good immunogenicity; rSjannexin immune rabbit polyclonal antibody titer reached 1:256000; female Sjannexin gene in schistosomula, higher transcription level in males and the paring adult sporocyst, eggs and miracidia, the transcription level is low, the transcription level of Sjannexin in the lowest cercariae; cercariae, schistosomula, female, expression the male and the paring adult, but not expressed in eggs; Sjannexin in Schistosoma japonicum male body; rSjannexin as antigen with biotin Pro And the amplification system (BAS-ELISA) detected from human serum, sensitivity and specificity were 80% and 90%; rSjannexin mice received 3.82% of worm reduction rate and liver egg reduction rate was 21.13%, the infection 40d ~ 42d stool egg reduction rate was 31.44%, 43D after infection to 45d faecal egg reduction rate 24.17%, but there were no differences in Statistics (P > 0.05).
Sj22 is a special protein of Schistosoma japonicum, containing 1 signal peptide; recombinant plasmid pET-28a/Sj22 was constructed, which induced expression of soluble protein by urea, purified recombinant protein was obtained with high concentration and purity; Western blot showed that rSj22 has certain immunogenicity free; rSj22 immune rabbit polyclonal antibody titer reached 1:256000 Sj22 gene; in females, higher transcription level and adult male from sporocysts, cercariae and miracidia, transcription level is low, and no transcription Sj22 in eggs; and in the form of adult male head, a small amount of expression in females, suggesting that Sj22 may be a sex related gene; immune localization show the expression of Sj22 in intestinal cavity of male Schistosoma japonicum, may be secreted protein, which is consistent with the Sj22 containing the signal peptide; rSj22 as antigen using avidin biotin amplification system (BAS-ELISA) detection The sensitivity and specificity of sera from different populations were 36.7% and 80%, respectively. Immunization with rSj22 immunized mice achieved 17.28% worm reduction rate and 9.03% liver egg reduction rate, and the fecal egg reduction rate of 43D to 45d was 22.53%, but there was no difference in statistical test (P > 0.05).
Sjinnexin of Schistosoma japonicum and important host homologous sequences in the lower similarity in the system; this study cloned and expressed the Sjinnexin of the second extracellular loop of prokaryotic expression (loop2, AA228 ~ AA331), obtained by urea soluble protein, purification, obtained with high concentration and purity of recombinant antigen fragment; Western blot showed that the animal serum infected with Schistosoma japonicum (rats and rabbits) and sera of patients with Schistosoma japonicum were higher than normal serum and animal serum have varying degrees of increase, indicating that it has good immunogenicity; Sjinnexin gene in sporocyst, miracidium, eggs, cercariae, schistosomula, both male and female, paring adult transcription, and the level is relatively stable; rSjinnexin immunized mice worm reduction rate of 12.21% (P > 0.05), liver egg reduction rate was 32.98% (P > 0.05), after infection 40d ~ 42d fecal egg reduction rate was 61.32% (P < 0.05), 43D after infection The fecal fecal reduction rate of ~ 45d is 53.55% (P > 0.05), indicating that it has a certain anti reproductive function and is a potential candidate target for anti reproductive vaccine.
SjMRLC with homologous sequences of Schistosoma japonicum in an important host in evolution belong to different branches, SjMRLC containing 1 EF-hand Ca~ (2+) binding domain, suggesting that it has Ca~ (2+) with the function; this study constructed pGEX-4T-3/SjMRLC recombinant plasmid, obtained soluble protein; SjMRLC gene in sporocysts, cercariae and miracidia, eggs., schistosomula, female, male and adult transcription were folded, and the level is relatively stable.

【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392.1

【引證文獻】

相關(guān)碩士學位論文 前1條

1 魏剛剛;血吸蟲蛋白抗原表位的預測及其在診斷抗原篩選方面的應用[D];華東理工大學;2013年

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本文編號：1356713