普氏立克次體主要蛋白抗原的重組表達和血清學(xué)分析
發(fā)布時間：2018-07-21 12:41
【摘要】：普氏立克次體(Rickettsia prowazekii)是流行性斑疹傷寒(epidemic typhus)的病原體。采用立克次體全菌抗原做間接免疫熒光(IFA)是最常用的實驗室血清學(xué)診斷斑疹傷寒方法,但是該IFA存在不同種屬立克次體菌體之間的抗體交叉反應(yīng),也影響該血清學(xué)診斷的可靠性。因此,發(fā)現(xiàn)普氏立克次體特異性蛋白抗原代替全菌抗原做流行性斑疹傷寒血清學(xué)診斷很有必要。 本研究選擇8個普氏立克次體主要蛋白抗原(EF-Ts、EF-Tu、FtsZ、 GroEL、Omp、Sca5、TolC、Rp828)作為流行性斑疹傷寒血清學(xué)診斷候選抗原。采用9對引物(其中一個基因分為2段擴增)做PCR,從普氏立克次體全基因組擴增9個目的基因片段。將擴增的9個基因片段與原核表達質(zhì)粒pET-32a(+)重組,再將重組質(zhì)粒分別轉(zhuǎn)化大腸埃希氏菌；在IPTG的誘導(dǎo)下9個轉(zhuǎn)化菌分別表達目的重組蛋白。采用鎳離子親和層析法(Ni-NTA)從轉(zhuǎn)化菌中純化這9個重組蛋白。 采用普氏立克次體、莫氏立克次體、立氏立克次體、黑龍江立克次體、恙蟲病東方體、貝氏柯克斯體感染小鼠血清與9個重組蛋白做免疫印跡,結(jié)果顯示FtsZ、GroEL、EF-Ts、Rp828等4個蛋白特異性最好,僅與普氏立克次體感染血清反應(yīng)。將9個重組蛋白點制一張微型蛋白芯片,再用立克次體感染血清分析蛋白芯片,結(jié)果顯示FtsZ、GroEL、Rp828、Sca5-1等4個蛋白呈現(xiàn)良好的血清學(xué)特異性,其僅與普氏立克次體和莫氏立克次體感染小鼠血清反應(yīng)陽性。我們研究結(jié)果證明FtsZ、Rp828、GroEL重組蛋白為斑疹傷寒立克次體的特異性抗原,其與Omp、EF-Ts、Sca5-1重組蛋白結(jié)合做血清學(xué)分析可顯著改善斑疹傷寒血清學(xué)診斷的特異性和敏感性。圖12幅,表3個,參考文獻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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前2條
1 高寧;溫博海;陳梅玲;牛東升;邱玲;;普氏立克次體120kDa表面蛋白N端和C端重組蛋白的抗原特異性研究[J];寄生蟲與醫(yī)學(xué)昆蟲學(xué)報;2006年02期
2 高寧,溫博海,魏文進,牛東升,邱玲,余躍飛,陳梅玲;普氏立克次體120kDa表面抗原N段基因的克隆與表達[J];中國人獸共患病雜志;2004年03期
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