【作者】 赵宏伟；

【导师】 高建民；

【作者基本信息】 福建师范大学 ， 发育生物学， 2005， 硕士

【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。更多还原

【Abstract】 DNase B is one of the secretory proteins of Group A 8-hemolytic streptococci (GAS), and exits in almost all GAS. DNase B sequence is conservative, can be used to test the antibody of DNase B. The anti-deoxyribonuclease-B titer (anti-DNase-B, or ADB) is regarded as one of the most sensitive markers of GAS infection, and recommended by Jones standard. However, the liquid culture of GAS is more complicated, dangerous and cost much. Furthermore, purifications of DNase B are complicated too. For these reasons, the gene for GAS DNase B has been cloned and vectors incorporating the cloned DNA have been used to transform Escherichia coli, allowing efficient and rapid production of the DNase B in E. coli without the necessity of growing large quantities of S. pyogenes.A strain of GAS was collected from a clinic sample. Primers were designed based on the published sequences of DNase B gene. The genomic DNA of GAS was isolated and used as the PCR template. The DNase B gene were amplified by PCR, the fragments of DNA were cloned into T-vector and checked by sequencing. The DNase B gene, which cloned in this experiment, has 98% homology with those loaded in Genebank.Another PCR were performed, the PCR productions were digested with endonuclease Kpnl and HindIII. and ligated with the plasmids pET-41a(+) cut by the same endonucleases, then transformed E.coli BL21(DE3). The recombinants were screened on the selected plates. One of the recombinants plasmids was isolated, identified, and sequenced. The fusion expressing vector of pET-41a (+) / DNase B was successfully constructed. The DNase B protein was expressed in E coli as fusion protein with glutathione Stransferase(GST) induced by IPTG. The fusion protein was purified by Ni-NTA column. Purified fusion protein was obtained. SDS-PAGE indicates: the recombinant protein mostly was soluble, and a little portion exited in inclusion body. The supernant flows through Ni-NTA gel for affinity chromatography, and thepurity is over 91.5%. The result of Western-blotting indicated the recombinant GST-DNase B fusion protein could specificly react with serum from GAS sufferer; there was an obvious band at 55KD. ELISA test confirms the specificity of this fusion protein. These results indicate that the fusion protein possesses favorable immuno-reactivity. So, these work have laid a solid foundation for the test of anti-DNase B and the research of GAS vaccine.更多还原