本文選題：A族溶血性鏈球菌　切入點(diǎn)：DNaseB　出處：《福建師范大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：DNase B是A族溶血性鏈球菌的一種分泌蛋白,序列高度保守,幾 乎存在于所有A族β-溶血性鏈球菌中,可用于抗DNA酶B的檢測(cè)。抗 DNA酶B檢測(cè)是Jones標(biāo)準(zhǔn)所推薦的鏈球菌感染及其并發(fā)癥的兩種實(shí)驗(yàn) 診斷方法之一。然而,A族β-溶血性鏈球菌的液體培養(yǎng)需要微需氧條件, 而且存在高致病性,純化天然DNase B危險(xiǎn)大,工藝復(fù)雜,成本高,不 利于大量臨床檢測(cè)的開展。為此,本實(shí)驗(yàn)克隆了DNase B基因,構(gòu)建了 一個(gè)原核表達(dá)載體,并且成功的在大腸桿菌中表達(dá)了這個(gè)蛋白。 本實(shí)驗(yàn)通過PCR方法擴(kuò)增出DNase B基因,連接入T載體,測(cè)序結(jié) 果證實(shí)序列正確。再次PCR,用KpnI、HindIII雙酶切PCR產(chǎn)物,后與 同樣酶切的pET-41a(+)連接,轉(zhuǎn)化大腸桿菌BL21(DE3)。提取轉(zhuǎn)化子質(zhì) 粒,菌落PCR及酶切鑒定證實(shí)DNase B基因片段插入表達(dá)載體,測(cè)序結(jié) 果表明本實(shí)驗(yàn)所克隆的DNase B基因與GenBank中登錄的基因序列同源 性在98%以上。表達(dá)菌用1mmol/L的IPTG誘導(dǎo)4小時(shí),收集菌體,破 菌,離心,分離上清和包涵體。SDS-PAGE顯示,目的蛋白主要以可溶蛋 白的形式存在,部分以包涵體形式存在。上清經(jīng)Ni-NTA凝膠親和層析, 純度達(dá)到91.5%以上。Western-blotting結(jié)果顯示,重組的GST-DNase B 融合蛋白能夠特異的與GAS感染患者的陽性血清反應(yīng),在55KD處有一 條明顯的條帶。ELISA實(shí)驗(yàn)顯示重組蛋白的抗原特異性良好。說明本實(shí) 驗(yàn)表達(dá)的融合蛋白的具有良好的免疫反應(yīng)性。為抗DNase B抗體的檢測(cè) 及疫苗研制打下堅(jiān)實(shí)的基礎(chǔ)。
[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.
【學(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：Q78

【引證文獻(xiàn)】

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本文編號(hào)：1635302