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Recombinant Expression and Serological Analyses of Major Protein Antigens of Rickettesia Prowazekii

Author: JiaYinJun
Tutor: WenBoHai
School: Central South University
Course: Biology
Keywords: Rickettsia prowazekii typhus major protein antigenrecombinant protein immunoblotting protein microarray
CLC: R392
Type: Master's thesis
Year: 2013
Downloads: 10
Quote: 0
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Abstract


Rickettsia prowazekii, the causative agent of epidemic typhus. The laboratory diagnosis of epidemic typhus is relied on indirect immunofluorescence assay (IFA) with rickettsial whole cells antigens (WCA), but serological cross-reactivity among various species WCAs affects its accuracy. Therefore, finding of species-specific antigens for serological diagnosis of epidemic typhus is necessary.In this study, eight major protein antigens (EF-Ts, EF-Tu, FtsZ, GroEL, Omp, Sca5, TolC, and Rp828) of R. prowazekii were chosen as candidate of serological diagnostic antigens for epidemic typhus. Eight genes were amplified with9pairs of primers separately (one gene was divided into two fragments to amplify). The amplified9gene fragments were linked with a expression plasmid pET-32a (+) and the resulting recombinant plasmids were used to transform Escherichia coli cells to express recombinant proteins after IPTG induction, respectively.Nine recombinant proteins purified from E. coli cells by Nickel affinity chromatography were immunoblotted with the sera from BALB/c mice experimentally infected with R. prowazekii, R. mooseri, R. rickettsii, R. heilongjiangensis, Orientia tsutsugamushi, or Coxiella burnetii separately. As a result, FtsZ, GroEL, EF-Ts, and Rp828reacted only with the sera from R. prowazeki i-infected mice. The nine recombinant proteins were fabricated a small protein microarray and the microarray was probed with these sera from mice infected with the various rickettsia. The result showed that FtsZ, GroEL, Rp828, and Sca5-1reacted positively only with the sera from mice infected with R. prowazekii or R. mooseri. Therefore, FtsZ、Rp828and GroEL are high specific to typhus group rickettsiae and their combination with Omp, EF-Ts, and Sca5-1will greatly improve the specificity and sensitivity of serological diagnosis of typhus.

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CLC: > Medicine, health > Basic Medical > Medical Immunology
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