日本血吸虫体被蛋白作为诊断和疫苗候选抗原的研究

发布时间：2017-12-30 22:05

本文关键词：日本血吸虫体被蛋白作为诊断和疫苗候选抗原的研究　出处：《中国疾病预防控制中心》2009年硕士论文　论文类型：学位论文

【摘要】： 血吸虫病仍然是影响人类健康的重要寄生虫病。现有的免疫学诊断方法主要是用血吸虫成虫粗抗原或虫卵粗抗原检测抗体,材料来源受限,且不能区分现症感染和既往感染,特异性也有待进一步提高。血吸虫疫苗一直是研究热点,但目前仍无突破性进展。血吸虫体被是血吸虫和宿主相互作用的两个主要界面之一,在营养吸收和免疫逃避等多个方面发挥重要作用,被认为是诊断、疫苗和药物的潜在靶点。前期日本血吸虫的体被蛋白质组学研究鉴定了一批体被蛋白质。本研究就是针对其中的Sjannexin(AY813612,AAW25344)、Sj22(AY815492,AAW27224)、Sjinnexin(AY815599,AAW27331)、SjMRLC(AY815219,AAW26951)等4个蛋白质开展相关研究,主要研究内容包括:(1)利用已知的序列进行生物信息学分析,如:信号肽预测、跨膜区域预测、功能位点和结构域预测、同源序列搜索、多序列比对和进化树构建等;(2)基因的原核克隆和表达、重组蛋白质的纯化;(3)重组蛋白免疫原性检测,重组蛋白兔多克隆抗体的制备及评价,基因的虫期转录特异性,蛋白质的虫期表达特异性,蛋白质的免疫定位;(4)蛋白质的诊断价值评价和蛋白质的动物保护性实验。 Sjannexin是相对保守的蛋白,与日本血吸虫重要宿主中的同源序列相似性较高,含有4个annexin重复单位结构域;构建了pET-28a/Sjannexin重组质粒,诱导表达获得可溶性蛋白,通过含咪唑的洗脱液纯化,获得了较高浓度和纯度的重组蛋白;Western blot检测表明rSjannexin具有较好的免疫原性;rSjannexin免疫兔多克隆抗体的滴度达到1:256,000;Sjannexin基因在童虫、雌虫、雄虫和合抱成虫中转录水平较高,胞蚴、毛蚴、虫卵中转录水平较低,尾蚴中转录水平最低;Sjannexin主要在尾蚴、童虫、雌虫、雄虫和合抱成虫中表达,而在虫卵中不表达;Sjannexin定位于日本血吸虫雄虫的体被;rSjannexin作为包被抗原,采用生物素亲和素放大系统(BAS-ELISA)检测不同人群血清,敏感度和特异度分别为80%和90%;用rSjannexin免疫小鼠获得了3.82%的减虫率,肝脏减卵率为21.13%,感染后40d～42d粪便减卵率为31.44%,感染后43d～45d粪便减卵率为24.17%,但统计学检验均无差异(p＞0.05)。 Sj22是日本血吸虫的特有蛋白,含有1个信号肽;构建了pET-28a/Sj22重组质粒,诱导表达获得不可溶性蛋白,通过尿素纯化,获得了较高浓度和纯度的重组蛋白;Western blot检测显示rSj22具有一定的免疫原性;rSj22免疫兔多克隆抗体的滴度达到1:256,000;Sj22基因在雌虫、雄虫和合抱成虫中转录水平较高,胞蚴、毛蚴、尾蚴和童虫中转录水平较低,而虫卵中无转录;Sj22主要在雄虫和合抱成虫中表达,在雌虫期有少量表达,提示Sj22可能是性别相关基因;免疫定位显示Sj22表达于日本血吸虫雄虫的肠腔,可能是分泌蛋白,这与Sj22含有信号肽相一致;rSj22作为包被抗原,采用生物素亲和素放大系统(BAS-ELISA)检测不同人群血清,敏感度和特异度分别为36.7%和80%;用rSj22免疫小鼠获得了17.28%的减虫率和9.03%的肝脏减卵率,感染后43d～45d粪便减卵率为22.53%,但统计学检验均无差异(p＞0.05)。 Sjinnexin与日本血吸虫重要宿主中的同源序列的相似性较低;本研究在原核表达系统中克隆并表达了Sjinnexin的第二个胞外环(loop2,AA228～AA331),获得不可溶性蛋白,通过尿素纯化,获得了较高浓度和纯度重组抗原片段;Western blot检测显示日本血吸虫感染动物血清(鼠和兔)及日本血吸虫患者血清均较正常动物血清和人血清有不同程度增强,表明其具有较好的免疫原性;Sjinnexin基因在胞蚴、毛蚴、虫卵、尾蚴、童虫、雌虫、雄虫和合抱成虫中均有转录,且水平比较稳定;用rSjinnexin免疫小鼠获得了12.21%的减虫率(p＞0.05),肝脏减卵率为32.98%(p＞0.05),感染后40d～42d粪便减卵率为61.32%(p＜0.05),感染后43d～45d粪便减卵率为53.55%(p＞0.05),说明其具有一定的抗生殖作用,是潜在的抗生殖疫苗候选靶标。 SjMRLC同日本血吸虫重要宿主中的同源序列在进化上属于不同的分支,SjMRLC含有1个EF-hand Ca~(2+)结合结构域,提示其具有Ca~(2+)结合功能;本研究构建了pGEX-4T-3/SjMRLC重组质粒,获得可溶性蛋白;SjMRLC基因在胞蚴、毛蚴、虫卵、尾蚴、童虫、雌虫、雄虫和合抱成虫中均有转录,且水平比较稳定。
[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.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

【引证文献】