【作者】 卞茂红；

【导师】 沈际佳； 沈继龙； 汪学龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2002， 硕士

【摘要】 目的：利用免疫学方法筛选日本血吸虫童虫cDNA文库，以寻找新的编码血吸虫病诊断和候选疫苗分子基因。方法：收集感染血吸虫患者混合血清，并对血清经滤膜吸附法以去除大肠杆菌抗体，以筛选日本血吸虫肝期章虫cDNA文库。从3次复筛得到的阳性克隆中选取3个进行插入片段的核苷酸序列测定，结果送GeneBank进行同源性分析。根据序列测定结果，选取其中一个具有完整开放阅读框架的基因，设计合成引物，PCR体外进行扩增，并将其克隆入pGEM-T载体，然后在表达载体pBK-CMV中亚克隆。用IPTG诱导后SDS-PAGE分析其表达，并用Westernblotting进行鉴定。结果：用抗血清初筛了约7.2×10~3个重组的噬菌体，得到48个阳性克隆，对其中的18个克隆进行三次复筛，得到12个持续阳性反应克隆。选取其中三个克隆进行测序并经同源性分析，结果其中编号为H4克隆的插入序列为编码日本血吸虫的14-3-3基因；编号为H9克隆的插入序列为编码日本血吸虫的线粒体基因；编号为H1克隆的插入序列长度为1186bp，与现有的基因无任何同源性，且具有一个591bp的完整开放阅读框，利用PCR法扩增该基因，克隆入载体pGEM-T，再亚克隆入表达载体PBK-CMV中，所形成的质粒经双酶切和以质粒为模板进行PCR扩增，均可获得一条与PCR产物一致的DNA片段。IPTG诱导后进行SDS-PAGE，发现在约26.5KDa位置出现一条表达条带，且随着表达时间的推移，表达产物量增大。Western blotting鉴定结果表明阳性病人的血清可特异性地识别此条带。结论：从日本血吸虫童虫cDNA文库中免疫筛选到一批编码对血吸虫的感染可能具有保护作用的分子，通过序列测定、克隆、诱导表达和鉴定，为进一步利用分子生物学方法研制其防治的疫苗和诊断抗原奠定基础。更多还原

【Abstract】 Objective: Schistosomulum japonicum cDNA Library was immunoscreened to discover genes encoding the novel molecules of diagnosis and candidate vaccine for schistosomiasis Memods:The mixture patient sera of schistosomiasis were collected and eliminated anti-E-coli antibodies by E-coli lysate,used as antibody to screen cDNA Library constructed.The 3 clones were selected from rescreening three times positive clones and followed sequencing.The obtained genes were compared against GeneBank datebase by BLASTN and BLASTP.Specific primers were designed based upon the sequences of insert which have complete open reading frame. The DNA was amplified using the plasmid as template.The product from PCR was cloned into pGEM-T vector.Then the target gene was subcloned into the expression vector(pBK-CMV).The recombinant vector was identified by restriction analysis and transformed into E.coliBL21.The expression was induced by IPTG and the result was identified using SDS-PAGE and Western blotting. Results: About 7.2 X 103 recombinant phages were screened,forty-eight positive clones were obtained in which eighteen were rescreened until the twelve continuing positive clones were comfirmed.The sequence data of three positive clones were analyzed in GeneBank datebase.Sequence of H4 clone is 5)14-3-3 gene and that of H9 clone is Sj mitochondrion gene. Sequence of insert of HI clone has 1186bp and a 591bp-size complete open reading frame(ORF). The HI DNA fragment was amplified by PCR and cloned into pGEM-T vector and then subcloned into pBK-CMV vector.The same bands were obtained by double restriction of recombinant plasmids and PCR using recombinant plasmids as template.After inducing by IPTG, the result of SDS-PAGE showed that the fusion protein was approximately 26.5KDa andgot more with time prolonging. Western blotting indentified that the band was specially recognized by sera from schistosomiasis patients infected. Conclusion: Some molecules coding possibly protective immunity for schistosomiasis were obtained by immunoscreening Schistosomulum japonicum cDNA Library. The following development of protective vaccine and diagnostic antigen was base on sequencing, cloning, inducing expressing and identifying by methods of molecule biology更多还原