日本血吸虫文库筛

发布时间：2018-04-14 21:32

本文选题：日本血吸虫 + 童虫　；参考：《安徽医科大学》2009年硕士论文

【摘要】： 目的筛选新的可能具有诊断意义的血吸虫蛋白分子,并对其进行生物信息学分析。方法以血吸虫病人血清筛选日本血吸虫15d肝期童虫cDNA文库,对阳性克隆插入片段的核苷酸进行序列分析,将结果通过Internet送入NCBI GenBank进行同源性比较并利用蛋白质分析软件进行生物信息学分析。结果经3轮筛选,共获15个阳性克隆,对其进行测序,获得11个基因,其中5个为编码日本血吸虫线粒体的基因,1个为编码日本血吸虫肌球蛋白的基因,其他5个分别为编码SjCHGC05315、SjCHGC01371、SjCHGC04782、SjCHGC05166、SjCHGC09769的基因。结论从日本血吸虫肝期童虫cDNA文库中筛选到一批日本血吸虫基因,为血吸虫病新的诊断候选分子的研究提供了材料。目的日本血吸虫病是中国严重的寄生虫病之一,政府每年投入大量资金用于血吸虫病的防治工作,但由于血吸虫生活史的复杂性,血吸虫病仍然没有得到很好的控制。血吸虫病控制的重要措施之一在于建立准确而又灵敏的诊断方法。本研究以寻找新的血吸虫病免疫诊断候选抗原分子为目的,对日本血吸虫表膜蛋白Tetraspanin2(SjTsp2)进行体外重组表达、纯化,建立重组抗原间接ELISA方法(rSj-ELISA)检测日本血吸虫重组表膜蛋白rSjTsp2用于日本血吸虫病免疫诊断的潜能,并与成虫粗抗原(SjAWA)平行检测,比较两种方法的敏感性和特异性之间的差异。并用纯化的重组蛋白rSjTsp2免疫小鼠,制备单克隆抗体,以进一步探讨rSjTsp2用于血吸虫病诊断的应用价值。方法诱导含SjTsp2编码基因的重组质粒pET28a(+)-SjTsp2表达出rSjTsp2,SDS-PAGE验证表达量,使用亲和层析法纯化重组蛋白,并用Lowry法检测纯化蛋白的浓度。将rSjTsp2和成虫粗抗原(SjAWA)包被反应板,棋盘滴定法分别确定最适抗原包被量和血清稀释度,建立rSjTsp2-ELISA和AWA -ELISA检测特异性抗体的方法并进行比较。用rSj Tsp2-ELISA和AWA-ELISA两种方法平行检测正常家兔血清,感染日本血吸虫后2周、4周和6周家兔血清,比较两种方法之间敏感性和特异性。继之,确定最佳试验条件后建立rSj Tsp2-ELISA检测34例急性、31例慢性和25例肝吸虫染者、9例线虫感染者和49例正常对照者血清标本。以rSjTsp2免疫小鼠6周后将其脾细胞与SP2/0骨髓瘤细胞进行杂交融合,HAT筛选出杂交瘤细胞,经亚克隆及扩大培养,获得5株单克隆抗体。用Antibody Isotype Kit鉴定单抗Ig类别及亚型,用间接ELISA法检测杂交瘤上清和小鼠腹水效价,免疫荧光实验鉴定其特异性。结果成功诱导表达重组质粒pET28a(+)-SjTsp2,6×His亲和层析柱纯化获得rSjTsp2,SDS-PAGE检测其分子量与理论值一致。以rSjTsp2作为诊断抗原分子,经过条件优化建立了rSjTsp2-ELISA诊断兔日本血吸虫病的方法。用rSjTsp2-ELISA,AWA-ELISA两种方法平行检测正常兔血清,感染日本血吸虫后2周、4周和6周兔血清,结果示rSjTsp2用于日本血吸虫病免疫诊断与AWA-ELISA具有相似的敏感性和特异性, rSjTsp2检测感染日本血吸虫后2周兔血清阳性率为75.4%,高于后者的68.9%。rSjTsp2检测血吸虫感染病人、其他寄生虫感染病人及正常人血清特异性抗体,初步结果显示该方法具有一定的敏感性和特异性。同时获得5株特异性抗rSjTsp2表膜蛋白的单克隆抗体的杂交瘤细胞株。单抗Ig类别及亚型,杂交瘤上清和小鼠腹水效价,免疫荧光鉴定其特异性,结果显示rSjTsp2为日本血吸虫成虫表膜蛋白。结论体外成功表达、纯化了日本血吸虫表膜蛋白Tetraspanin2(SjTsp2),建立了间接rSj-ELISA方法检测感染家兔血清IgG抗体,其原核表达重组蛋白具有一定的诊断应用潜能。本研究还获得了5株特异性抗rSjTsp2表膜蛋白的单克隆抗体的杂交瘤细胞株。
[Abstract]:Objective to screen new schistosomiasis protein molecules that may have diagnostic significance and to carry out bioinformatics analysis.
Methods schistosomiasis screening of Schistosoma japonicum 15d hepatic schistosomula cDNA library, the positive clone fragment of nucleotide sequence analysis results through the Internet into the NCBI GenBank homology and protein analysis software by bioinformatic analysis.
Results after 3 rounds of screening, 15 positive clones were obtained and sequenced to obtain 11 genes, 5 of which encoding Schistosoma japonicum mitochondrial genes, 1 genes encoding for Schistosoma japonicum tropomyosin, the other 5 were SjCHGC01371, encoding SjCHGC05315, SjCHGC04782, SjCHGC05166, SjCHGC09769 genes.
Conclusion a number of Schistosoma japonicum genes were screened from Schistosoma japonicum liver worm cDNA library, providing materials for the study of new diagnostic candidates for schistosomiasis.
Objective schistosomiasis is one of the most serious parasitic disease China, the government invested a lot of money every year for schistosomiasis prevention and control work, but because of the complexity of the life history of schistosomiasis japonicum, still has not been well controlled. One of the important measures for schistosomiasis control is to establish accurate and sensitive diagnostic methods. This study to find the candidate antigen for immunodiagnosis of schistosomiasis new molecules for the purpose of Schistosoma japonicum membrane protein Tetraspanin2 (SjTsp2). The recombinant expression, purification and establishment of indirect ELISA recombinant antigen (rSj-ELISA) detection of Schistosoma japonicum recombinant membrane protein rSjTsp2 for immunodiagnosis of schistosomiasis japonica potential, and adult worm antigen (SjAWA) parallel detection, the difference between the two the sensitivity and specificity of the methods. And immunized with rSjTsp2 recombinant protein were purified, the preparation of single To clone antibody to further explore the application value of rSjTsp2 in the diagnosis of schistosomiasis.
The recombinant plasmid pET28a containing SjTsp2 gene encoding method to induce the expression of -SjTsp2 (+) rSjTsp2, expression of SDS-PAGE authentication, using the recombinant protein was purified by affinity chromatography, and the concentration of purified protein was detected by Lowry. RSjTsp2 and adult worm antigen (SjAWA) coated reaction plate, checkerboard titration method to determine the optimal antigen and the amount of serum dilution, and compared the method of establishing rSjTsp2-ELISA and AWA -ELISA detection of specific antibody. The parallel detection of normal rabbit serum by rSj Tsp2-ELISA and AWA-ELISA two methods, 2 weeks of infection of Schistosoma japonicum after 4 weeks and 6 weeks of rabbit serum, sensitivity and specificity of the comparison between the two methods. Then, the establishment of rSj Tsp2-ELISA was detected in 34 cases of acute to determine the optimal experimental conditions, 31 cases and 25 cases of chronic liver fluke infections, serum samples from 9 cases of nematode infections and 49 cases of normal controls. In 6 weeks after immunization of mice will be rSjTsp2 The spleen cells and myeloma cell SP2/0 were screened by HAT fusion, hybridoma cells, by subcloning andexpansion of training, obtained 5 monoclonal antibodies. Using Antibody Isotype Kit identification of monoclonal antibody Ig type and subtype detection, hybridoma supernatant and ascites titer by indirect ELISA, immunofluorescence assay. The opposite sex.
The expression of recombinant plasmid pET28a (+) rSjTsp2 affinity chromatography purification was -SjTsp2,6 * His, SDS-PAGE was consistent with the molecular weight and theoretical value. Using rSjTsp2 as diagnostic antigen molecules, after optimizing the condition to establish a diagnosis of rSjTsp2-ELISA rabbit schistosomiasis method. RSjTsp2-ELISA AWA-ELISA two parallel detection method of normal rabbit serum. The infection of Schistosoma japonicum after 2 weeks, 4 weeks and 6 weeks of rabbit serum, the results showed that rSjTsp2 in Japan for the immunodiagnosis of schistosomiasis and AWA-ELISA have similar sensitivity and specificity of rSjTsp2 detection on Schistosoma japonicum infection after 2 weeks of rabbit serum positive rate was 75.4%, higher than that in patients with 68.9%.rSjTsp2 infection the detection of schistosome, patients and normal human serum. The specific antibody infected with other parasites, preliminary results show that this method has certain sensitivity and specificity. At the same time obtain 5 strains of specific anti rSj The hybridoma cell lines with monoclonal antibodies against Tsp2 membrane proteins. Ig and its subtypes, the hybridoma supernatants and the ascites of mice were identified by immunofluorescence. The results showed that rSjTsp2 is the membrane protein of Schistosoma japonicum adult.
Conclusion in vitro successfully expressed Schistosoma japonicum membrane purified protein Tetraspanin2 (SjTsp2), was developed for the detection of serum IgG antibody in rabbits infected with indirect rSj-ELISA and prokaryotic expression of the recombinant protein has certain diagnostic potential. This study also obtained hybridoma cell lines of monoclonal antibody specific against 5 strains of the rSjTsp2 membrane protein.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R383.2

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