DNase B基因的克隆及在大肠杆菌中的表达

发布时间：2018-03-19 17:35

本文选题：A族溶血性链球菌　切入点：DNaseB　出处：《福建师范大学》2005年硕士论文　论文类型：学位论文

【摘要】：DNase B是A族溶血性链球菌的一种分泌蛋白,序列高度保守,几乎存在于所有A族β-溶血性链球菌中,可用于抗DNA酶B的检测。抗 DNA酶B检测是Jones标准所推荐的链球菌感染及其并发症的两种实验诊断方法之一。然而,A族β-溶血性链球菌的液体培养需要微需氧条件, 而且存在高致病性,纯化天然DNase B危险大,工艺复杂,成本高,不利于大量临床检测的开展。为此,本实验克隆了DNase B基因,构建了一个原核表达载体,并且成功的在大肠杆菌中表达了这个蛋白。本实验通过PCR方法扩增出DNase B基因,连接入T载体,测序结果证实序列正确。再次PCR,用KpnI、HindIII双酶切PCR产物,后与同样酶切的pET-41a(+)连接,转化大肠杆菌BL21(DE3)。提取转化子质粒,菌落PCR及酶切鉴定证实DNase B基因片段插入表达载体,测序结果表明本实验所克隆的DNase B基因与GenBank中登录的基因序列同源性在98%以上。表达菌用1mmol/L的IPTG诱导4小时,收集菌体,破菌,离心,分离上清和包涵体。SDS-PAGE显示,目的蛋白主要以可溶蛋白的形式存在,部分以包涵体形式存在。上清经Ni-NTA凝胶亲和层析, 纯度达到91.5%以上。Western-blotting结果显示,重组的GST-DNase B 融合蛋白能够特异的与GAS感染患者的阳性血清反应,在55KD处有一条明显的条带。ELISA实验显示重组蛋白的抗原特异性良好。说明本实验表达的融合蛋白的具有良好的免疫反应性。为抗DNase B抗体的检测及疫苗研制打下坚实的基础。
[Abstract]:DNase B is a secretory protein of group A hemolytic streptococcus, highly conserved. It is found in all group A 尾 -hemolytic streptococcus and can be used to detect DNA enzyme B. Detection of DNA enzyme B is two kinds of experiments of streptococcus infection and its complications recommended by Jones standard. One of the diagnostic methods. However, liquid culture of Group A 尾 -hemolytic Streptococcus requires microaerobic conditions. Moreover, there is high pathogenicity, the purification of natural DNase B is dangerous, the process is complex, the cost is high, Therefore, DNase B gene was cloned and constructed. A prokaryotic expression vector and successfully expressed the protein in Escherichia coli. In this experiment, DNase B gene was amplified by PCR, ligated into T vector and sequenced. It was confirmed that the sequence was correct. Once again, the PCR product was digested with KpnI HindIII double enzyme. The same enzyme cleavage pET-41a () was ligated and transformed into Escherichia coli BL21DE3. The transformant was extracted. Granulocyte, colony PCR and restriction endonuclease analysis confirmed that DNase B gene fragment was inserted into expression vector and sequenced. The results show that the cloned DNase B gene is homologous to the GenBank gene sequence. The expression bacteria were induced with 1 mmol / L IPTG for 4 hours. Bacteria, centrifugation, separation of supernatant and inclusion body. SDS-PAGE showed that the target protein was mainly soluble egg. The supernatant exists in the form of inclusion body. The supernatant is subjected to Ni-NTA gel affinity chromatography. The purity was more than 91.5%. Western-blotting results showed that the recombinant GST-DNase B. The fusion protein can specifically react with the positive serum of patients with GAS infection. The results of Elisa showed that the antigen specificity of the recombinant protein was good. The expressed fusion protein has good immunoreactivity. Detection of anti DNase B antibody. And vaccine development lay a solid foundation.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78

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