弓形虫缓殖子期特异抗原BSR4的克

发布时间：2018-05-07 15:21

本文选题：弓形虫 + BSR4　；参考：《蚌埠医学院》2011年硕士论文

【摘要】：目的从弓形虫Prugniaud（PRU）株总DNA中克隆BSR4基因片段，构建pET28a(+)-BSR4重组表达质粒；对比不同虫株之间BSR4基因序列，进行生物信息学分析；在原核表达系统中表达BSR4，纯化表达产物并检测其免疫反应性，为弓形虫病的免疫诊断和疫苗研制以及致病机制的研究奠定基础。方法用本实验室保种的弓形虫PRU株腹腔接种昆明鼠，取脑组织匀浆，用小鼠淋巴细胞分离液纯化弓形虫包囊，提取弓形虫PRU株的基因组DNA，根据GenBank中已知弓形虫ME49株BSR4基因序列设计合成引物，在引物中引入NcoI和HindⅢ酶切位点。应用PCR技术从弓形虫PRU株基因组DNA中扩增BSR4基因。PCR产物经胶回收纯化后用限制性内切酶NcoⅠ和HindⅢ切下目的片段，以相同的酶切位点通过T4连接酶亚克隆入pET28a(+)载体中构建重组表达质粒pET28a(+)-BSR4，采用双酶切及测序方法鉴定重组质粒。采用生物信息学方法对BSR4蛋白的理化性质、同源性分析以及二级结构和抗原表位进行预测。将重组质粒pET28a(+)-BSR4转化至表达宿主菌BL21(DE3)中，挑取单个菌落，培养扩增至OD600约为0.5，加IPTG诱导表达，超声破壁后，分离上清和沉淀，SDS-PAGE电泳分析表达产物，对沉淀中的包涵体经8M尿素溶解，，His Trap Ni亲和层析柱纯化重组蛋白，Western-Blottting鉴定重组BSR4蛋白的免疫反应性。改良Bradford(考马斯亮蓝)法测定纯化蛋白浓度。用小鼠淋巴细胞分离液分离感染及未感染弓形虫小鼠脾细胞，以纯化抗原刺激小鼠脾细胞的生长，48h后加入3H-TdR，继续培养24h，检测小鼠脾细胞淋巴细胞增殖水平。结果PCR体外扩增得到目的基因片段长约1194bp，测序结果表明，获得弓形虫BSR4基因片段。经BLAST同源性对比显示，弓形虫PRU株和ME49株基因序列同源性为100%，是高度保守的序列。双酶切及测序结果显示目的基因己被正确地连接入重组质粒pET28a(+)中。生物信息学分析表明，BSR4蛋白相对分子量为42344.75，有2个功能结构域，氨基酸序列的前40位为信号肽序列，N端为信号肽，C端为疏水序列，预测它为糖基磷脂酰肌醇固着蛋白,存在18个潜在抗原表位及2个保守功能区域。重组质粒pET28a(+)-BSR4经IPTG诱导在大肠埃希菌BL21(DE3)中表达，超声破菌后SDS-PAGE电泳分析取上清和沉淀，结果表明重组蛋白在37℃时大量表达，以包涵体的形式存在。表达蛋白经尿素溶解，His Trap Ni亲和层析柱纯化，得到可溶性蛋白。Western-blotting检测可发现一特异性蛋白区带，分子量约为45kDa，该条带能被弓形虫慢性感染血清识别。淋巴细胞增殖实验表明，感染弓形虫小鼠刺激指数（SI）明显高于未感染组。结论从弓形虫PRU株中克隆出缓殖子期特异性抗原BSR4基因编码序列；成功构建了目的基因BSR4的重组质粒pET28a(+)-BSR4，在大肠埃希菌BL21中以包涵体形式高效表达了BSR4目的基因片段；该产物具有特异的免疫反应性，在体外可刺激记忆性淋巴细胞明显增殖。推测BSR4可能是具有良好应用前景的弓形虫候选疫苗分子，还有望应用于弓形虫慢性感染的检测。
[Abstract]:Objective To clone BSR4 gene fragment from the total DNA of Toxoplasma gondii ( PRU ) strain and construct pET28a ( + ) - BSR4 recombinant expression plasmid ;
Bioinformatic analysis was carried out against the sequence of BSR4 gene between different insect strains .
BSR4 was expressed in the prokaryotic expression system , the expression product was purified and its immunoreactivity was detected , which laid the foundation for the study of the immunological diagnosis and vaccine development and the pathogenesis of toxoplasmosis .

Methods The recombinant plasmid pET28a ( + ) - BSR4 was isolated from the genomic DNA of Toxoplasma gondii by PCR . The recombinant plasmid pET28a ( + ) - BSR4 was isolated from the genomic DNA of Toxoplasma gondii . The recombinant plasmid pET28a ( + ) - BSR4 was isolated and purified . The recombinant plasmid pET28a ( + ) - BSR4 was isolated by PCR .

coli BL21 ( DE3 ) . The recombinant plasmid pET28a ( + ) - BSR4 was purified by IPTG to obtain soluble protein . The recombinant plasmid pET28a ( + ) - BSR4 was purified by IPTG to obtain soluble protein . The recombinant plasmid pET28a ( + ) - BSR4 was purified by IPTG to obtain soluble protein .

Conclusion BSR4 gene coding sequence is cloned from the PRU strain of Toxoplasma gondii .
The recombinant plasmid pET28a ( + ) - BSR4 of the target gene BSR4 was successfully constructed , and the gene fragment of BSR4 was expressed in the form of inclusion bodies in E . coli BL21 .
The product has specific immunoreactivity and can stimulate memory lymphocyte proliferation in vitro . It is speculated that BSR4 may be a candidate for toxoplasmosis with good application prospect . It is also expected to be applied to the detection of chronic infection of Toxoplasma gondii .

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【参考文献】