弓形虫表面抗原SAG3和SAG1基因的克隆表达、纯化及鉴定

发布时间：2018-05-26 20:06

本文选题：弓形虫 + SAG3　；参考：《广西医科大学》2010年硕士论文

【摘要】： 目的:运用PCR体外扩增法获取弓形虫表面抗原SAG3和SAG1目的基因片段,构建重组表达质粒pET29a(+)-SAG3和pET29a(+)-SAG1,分别在大肠埃希菌(Escherichia coli)中诱导表达SAG3和SAG1重组蛋白,利用镍离子柱分离纯化重组蛋白,蛋白印迹(Western blotting)检测重组蛋白的抗原性,为弓形虫病的混合多价蛋白疫苗和核酸疫苗研制奠定基础。方法:从RH株弓形虫保种小鼠腹水中收集滋养体,用小量组织/细胞基因组DNA抽提试剂盒提取弓形虫基因组DNA。根据GenBank中弓形虫RH标准株的SAG3和SAG1基因序列,利用Primer5.0软件分别各设计一对特异性引物,以基因组DNA为模板,进行体外PCR扩增,产物经琼脂糖凝胶电泳、测序和BLAST同源比对分析鉴定,获得SAG3和SAG1基因片段。SAG3基因片段经Biospin胶回收试剂盒进行回收纯化,连接至pUCm-T载体中构建克隆质粒pUCm-T-SAG3,以菌落PCR、双酶切和测序方法鉴定,用限制性内切酶NdeⅠ、EcoRⅠ从pUCm-T-SAG3中切下SAG3基因片段,亚克隆至质粒pET29a(+)中;而SAG1基因片段回收纯化后则直接用限制性内切酶NdeⅠ和XhoⅠ双酶切后定向克隆至质粒pET29a(+)中。构建的重组表达质粒pET29a(+)-SAG3和pET29a(+)-SAG1均采用菌落PCR、双酶切和测序等方法进行鉴定。将重组表达质粒pET29a(+)-SAG3和pET29a(+)-SAG1分别转入E.coliBL21中,经IPTG诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白表达情况,并用SPSS软件对重组蛋白分子量进行计算分析。用小鼠Anti-His抗体作一抗经Western blotting鉴定重组蛋白的组氨酸标签。重组蛋白经镍离子柱纯化后,再用Western blotting分析纯化的重组蛋白与小鼠弓形虫阳性血清的免疫反应性。结果:用SAG3和SAG1特异性引物进行PCR体外扩增,产物经凝胶电泳和测序分析,分别获得长1 059bp和799 bp的基因片段,BLAST同源性分析显示两个目的片段与弓形虫RH株相对应的SAG3基因和SAG1基因的cDNA同源性为100%。克隆质粒pUCm-T-SAG3经鉴定,琼脂糖凝胶电泳显示在1 000bp以上处有特异性条带,测序结果也显示SAG3目的基因片段的已正确地插入克隆质粒T7启动子下游,并含有NdeⅠ、EcoRⅠ酶切位点。重组表达质粒pET29a(+)-SAG3和pET29a(+)-SAG1经菌落PCR和双酶切鉴定后琼脂糖凝胶电泳显示分别在1 000bp和800bp处得到与预期SAG3和SAG1目的基因片段大小一致的条带,测序鉴定后显示两个目的基因片段均插入重组表达质粒pET29a(+)序列T7启动子及核糖结合位点(rbs)AAGGAG序列的下游NdeⅠ酶切位点处,通过读码分析在重组蛋白末端能表达质粒自身所带的组氨酸标签。经IPTG诱导后,两个重组表达质粒均能在BL21中稳定表达重组蛋白,通过SDS-PAGE电泳检测到特异性蛋白表达条带,用直线相关分析得出重组蛋白SAG3和SAG1的相对分子量(Mr)分别为41 366.2和29 991.6。两种重组蛋白用Western blotting检测后均能与小鼠Anti-His抗体有特异性反应条带。用镍离子柱纯化后经SDS-PAGE电泳显示出两种重组蛋白均可得到背景清晰的纯化蛋白条带,纯化重组蛋白通过Western blotting检测,两种纯化重组蛋白能与小鼠弓形虫感染血清有特异反应条带。结论:用PCR方法成功从弓形虫RH株速殖子基因中扩增出长为1 059bp的SAG3和799 bp的SAG1基因片段。成功地构建重组表达质粒pET29a(+)-SAG3和pET29a(+)-SAG1,使弓形虫表面抗原SAG3和SAG1重组蛋白在体外获得表达,两种重组表达蛋白具有较好的活性。用镍离子柱成功纯化出重组蛋白SAG3和SAG1,两种纯化的重组蛋白亦显示出了良好的抗原性。以此推测出SAG3和SAG1基因作为弓形虫候选疫苗分子具有良好应用前景,纯化的重组SAG3和SAG1蛋白为弓形虫混合多价蛋白疫苗的研制奠定基础。
[Abstract]:Objective : To construct recombinant expression plasmid pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 by PCR in vitro amplification . The recombinant expression plasmid pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 were used to induce the expression of SAG3 and SAG1 in Escherichia coli . Western blotting was used to detect the antigenicity of recombinant protein .

The recombinant expression plasmid pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 were cloned into plasmid pET29a ( + ) . The recombinant expression plasmid pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 were cloned into plasmid pET29a ( + ) . The recombinant expression plasmid pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 were cloned into plasmid pET29a ( + ) .

Results : The gene fragments of SAG3 and SAG1 were amplified by SDS - PAGE . The results showed that the two recombinant proteins were inserted into the downstream of the recombinant expression plasmid pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 . The recombinant expression plasmid pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 were identified by Western blotting .

Conclusion : The SAG3 and SAG1 fragments of SAG3 and SAG1 were successfully constructed by PCR . The recombinant expression plasmids pET29a ( + ) - SAG3 and pET29a ( + ) - SAG1 were successfully constructed . The recombinant protein SAG3 and SAG1 were successfully expressed in vitro .
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R382.5

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