普氏立克次体主要蛋白抗原的重组表达和血清学分析
发布时间：2018-07-21 12:41
【摘要】：普氏立克次体(Rickettsia prowazekii)是流行性斑疹伤寒(epidemic typhus)的病原体。采用立克次体全菌抗原做间接免疫荧光(IFA)是最常用的实验室血清学诊断斑疹伤寒方法,但是该IFA存在不同种属立克次体菌体之间的抗体交叉反应,也影响该血清学诊断的可靠性。因此,发现普氏立克次体特异性蛋白抗原代替全菌抗原做流行性斑疹伤寒血清学诊断很有必要。 本研究选择8个普氏立克次体主要蛋白抗原(EF-Ts、EF-Tu、FtsZ、 GroEL、Omp、Sca5、TolC、Rp828)作为流行性斑疹伤寒血清学诊断候选抗原。采用9对引物(其中一个基因分为2段扩增)做PCR,从普氏立克次体全基因组扩增9个目的基因片段。将扩增的9个基因片段与原核表达质粒pET-32a(+)重组,再将重组质粒分别转化大肠埃希氏菌；在IPTG的诱导下9个转化菌分别表达目的重组蛋白。采用镍离子亲和层析法(Ni-NTA)从转化菌中纯化这9个重组蛋白。 采用普氏立克次体、莫氏立克次体、立氏立克次体、黑龙江立克次体、恙虫病东方体、贝氏柯克斯体感染小鼠血清与9个重组蛋白做免疫印迹,结果显示FtsZ、GroEL、EF-Ts、Rp828等4个蛋白特异性最好,仅与普氏立克次体感染血清反应。将9个重组蛋白点制一张微型蛋白芯片,再用立克次体感染血清分析蛋白芯片,结果显示FtsZ、GroEL、Rp828、Sca5-1等4个蛋白呈现良好的血清学特异性,其仅与普氏立克次体和莫氏立克次体感染小鼠血清反应阳性。我们研究结果证明FtsZ、Rp828、GroEL重组蛋白为斑疹伤寒立克次体的特异性抗原,其与Omp、EF-Ts、Sca5-1重组蛋白结合做血清学分析可显著改善斑疹伤寒血清学诊断的特异性和敏感性。图12幅,表3个,参考文献46篇。
[Abstract]:Rickettsia prowazekii is the pathogen of epidemic typhus (epidemic typhus). Indirect immunofluorescence (IFA) with Rickettsiella whole bacteria antigen is the most commonly used laboratory serological diagnosis method for typhus fever, but the IFA has antibody cross reaction between different species of Rickettsiella. It also affects the reliability of the serological diagnosis. Therefore, it is necessary to find the specific protein antigen of Rickettsia przewalskii to replace the whole bacterial antigen for the serological diagnosis of epidemic typhus fever. In this study, eight major protein antigens of Rickettsiella przewalskii (EF-TsTS-EF-Tu-FtsZ, GroELOmp-Sca5TolCp828) were selected as candidate antigens for the serological diagnosis of epidemic typhus. Nine target gene fragments were amplified from the whole genome of Rickettsia przewalskii by using 9 pairs of primers (one of which was divided into two segments). The amplified nine gene fragments were recombined with the prokaryotic expression plasmid pET-32a (), and the recombinant plasmids were transformed into Escherichia coli, respectively. The nine recombinant proteins were purified by nickel ion affinity chromatography (Ni-NTA) from the transformed bacteria. The sera of mice infected with Rickettsia przewalskii, Rickettsiella mori, Rickettsiella monocytogenes, Rickettsiella tsutsugamushi, Coxburella beersoni were imprinted with 9 recombinant proteins. The results showed that the four proteins of FtsZG GroELL EF-Tsfen Rp828 had the best specificity, and only reacted with the serum of Rickettsiella przewalskii infection. A microchip was prepared from 9 recombinant protein sites and then infected with Rickettsia. The results showed that FtsZG GroELL rp828 Sca5-1 and other four proteins showed good serological specificity. It was only positive in sera of mice infected with Rickettsia przewalskii and Rickettsia Morse. Our results showed that the recombinant protein of Rp828 / GroEL was a specific antigen of typhus, and the combination of FtsZH rp828 and OmpEF-Tsf- Sca5-1 protein could significantly improve the specificity and sensitivity of typhus serological diagnosis. 12 figures, 3 tables, 46 references.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392
本文编号：2135560
[Abstract]:Rickettsia prowazekii is the pathogen of epidemic typhus (epidemic typhus). Indirect immunofluorescence (IFA) with Rickettsiella whole bacteria antigen is the most commonly used laboratory serological diagnosis method for typhus fever, but the IFA has antibody cross reaction between different species of Rickettsiella. It also affects the reliability of the serological diagnosis. Therefore, it is necessary to find the specific protein antigen of Rickettsia przewalskii to replace the whole bacterial antigen for the serological diagnosis of epidemic typhus fever. In this study, eight major protein antigens of Rickettsiella przewalskii (EF-TsTS-EF-Tu-FtsZ, GroELOmp-Sca5TolCp828) were selected as candidate antigens for the serological diagnosis of epidemic typhus. Nine target gene fragments were amplified from the whole genome of Rickettsia przewalskii by using 9 pairs of primers (one of which was divided into two segments). The amplified nine gene fragments were recombined with the prokaryotic expression plasmid pET-32a (), and the recombinant plasmids were transformed into Escherichia coli, respectively. The nine recombinant proteins were purified by nickel ion affinity chromatography (Ni-NTA) from the transformed bacteria. The sera of mice infected with Rickettsia przewalskii, Rickettsiella mori, Rickettsiella monocytogenes, Rickettsiella tsutsugamushi, Coxburella beersoni were imprinted with 9 recombinant proteins. The results showed that the four proteins of FtsZG GroELL EF-Tsfen Rp828 had the best specificity, and only reacted with the serum of Rickettsiella przewalskii infection. A microchip was prepared from 9 recombinant protein sites and then infected with Rickettsia. The results showed that FtsZG GroELL rp828 Sca5-1 and other four proteins showed good serological specificity. It was only positive in sera of mice infected with Rickettsia przewalskii and Rickettsia Morse. Our results showed that the recombinant protein of Rp828 / GroEL was a specific antigen of typhus, and the combination of FtsZH rp828 and OmpEF-Tsf- Sca5-1 protein could significantly improve the specificity and sensitivity of typhus serological diagnosis. 12 figures, 3 tables, 46 references.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 高宁;温博海;陈梅玲;牛东升;邱玲;;普氏立克次体120kDa表面蛋白N端和C端重组蛋白的抗原特异性研究[J];寄生虫与医学昆虫学报;2006年02期
2 高宁,温博海,魏文进,牛东升,邱玲,余跃飞,陈梅玲;普氏立克次体120kDa表面抗原N段基因的克隆与表达[J];中国人兽共患病杂志;2004年03期
，本文编号：2135560
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