Evaluation of the recombinant protein Tp0965 of Treponema pallidum as candidate antigen for serological diagnosis of syphilis

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Abstract


Backgraund: Treponemal tests based on detection of antibodies against the T. pallidum antigens are the most specific methods for serological diagnosis of syphilis. Due to inability to cultivate this bacterium in vitro, the most promising sources of its antigens for diagnostic reactions are recombinant proteins of T. pallidum. Cloning and evaluation of the analytical value of certain T. pallidum proteins is the approach to improve the sensitivity, specificity, and reproducibility of serological tests for syphilis, including possibilities of differential diagnosis of various forms of the disease.

Objective: The aim of present study was to evaluate the analytical values (sensitivity and specificity) of Tp0965 recombinant protein of T. pallidum as a candidate antigen for serological diagnosis of syphilis.

Methods: Tp0965 gene was amplified by polymerase chain reaction from T. pallidum genomic DNA (Nichol's strain) and the nucleotide sequence was cloned into the expression vector pET28a. E. coli BL-21 (DE3) cells were transformed with this plasmid, and the recombinant protein production was induced by isopropyl-β-D-1-thiogalactopyranoside. Isolation and purification of Tp0965 recombinant protein from bacterial lysate was performed by metal‐chelate affinity chromatography using Ni-NTA Sepharose. The collected protein was seeded on high binding 96-well plates, which were used for ELISA with sera of patients with various forms of syphilis in comparison with healthy donors.

Results: High frequency of positive ELISA results was shown with serum of patients with syphilis, compared with a group of healthy donors. The sensitivity of serological reactions using recombinant protein Tr0965 was 98.8%, specificity - 87.5%. The highest sensitivity (100%) was detected in the groups of patients with primary, secondary and early latent syphilis, while in the group of patients with late latent syphilis it decreased to 95.2%.

Conclusions: We concluded that due to their specificity the recombinant protein Tp0965 T.pallidum can be used as a candidate antigen for development of syphilis serological diagnostic assays. 


A. V. Runina

State Research Center of Dermatovenerology and Cosmetology, Moscow

Email: bel4788@gmail.com

Russian Federation Researcher

A. S. Starovoitova

Lomonosov Moscow State University

Email: starovoitova.anastasiya@mail.ru

Russian Federation

Student

D. G. Deryabin

State Research Center of Dermatovenerology and Cosmetology, Moscow

Author for correspondence.
Email: dgderyabin@yandex.ru

Russian Federation MD, PhD. Professor, Head of Department

A. A. Kubanov

State Research Center of Dermatovenerology and Cosmetology, Moscow

Email: info@cnikvi.ru

Russian Federation MD, PhD. Professor, Deputy Director

  1. Brinkman MB, McKevitt M, McLoughlin M, et al. Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome. J Clin Microbiol. 2006;44(3):888– 891. doi: 10.1128/JCM.44.3.888-891.2006.
  2. Ratnam S. The laboratory diagnosis of syphilis. Can J Infect Dis Med Microbiol. 2005;16(1):45–51. doi: 10.1155/2005/597580.
  3. Carlson JA, Dabiri G, Cribier B, Sell S. The immunopathobiology of syphilis: the manifestations and course of syphilis are determined by the level of delayed-type hypersensitivity. Am J Dermatopathol. 2011;33(5):433–460. doi: 10.1097/DAD.0b013e3181e8b587.
  4. Соколовский Е., Фриго Н., Ротанов С., и др. Восточноевропейская система сексуального и репродуктивного здоровья. Руководство по лабораторной диагностике сифилиса в странах Восточной Европы // Вестник дерматологии и венерологии. — 2008. — №5. — С. 87—96. [Sokolovskii E, Frigo N, Rotanov S, et al. EE SRH Network. Guidelines for the laboratory diagnosis of syphilis in East-European countries. Vestn Dermatol Venerol. 2008;(5):87−96. (In Russ).]
  5. Smith BC, Simpson Y, Morshed MG, et al. New proteins for a new perspective on syphilis diagnosis. J Clin Microbiol. 2013;51(1):105– 111. doi: 10.1128/JCM.01390-12.
  6. Long FQ, Zhang JP, Shang GD, et al. Seroreactivity and immunogenicity of Tp0965, a hypothetical membrane protein of Treponema pallidum. Chin Med J (Engl). 2012;125(11):1920– 1924.
  7. Castro R, Prieto ES, Santo I, et al. Evaluation of an enzyme immunoassay technique for detection of antibodies against Treponema pallidum. J Clin Microbiol. 2003;41(1):250-253. doi: 10.1128/jcm.41.1.250-253.2003.
  8. Yu CS, Chen YC, Lu CH, Hwang JK. Prediction of protein subcellular localization. Proteins. 2006;64(3):643-651. doi: 10.1002/prot.21018.
  9. Zhang RL, Zhang JP, Wang QQ. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer. PLoS One. 2014;9(12):e115134. doi: 10.1371/journal.pone.0115134.
  10. Хайруллин Р.Ф., Ротанов С.В., Фриго Н.В., Белоусова А.В. Биоинформатический анализ специфических антигенов T. pallidum // Вестник дерматологии и венерологии. —2012. — №5. — С. 56—64. [Khairullin R, Rotanov S, Frigo N, Belousova A. Bioinformatic analysis of T. pallidum specific antigens. Vestn Dermatol Venerol. 2012;(5):56−64. (In Russ).]
  11. McGill MA, Edmondson DG, Carroll JA, et al. Characterization and serologic analysis of the Treponema pallidum proteome. Infect Immun. 2010;78(6):2631–2643. doi: 10.1128/IAI.00173-10.

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