FEASIBILITY TO USE OF RECOMBINANT DOMAINS OF IMMUNOGLOBULIN-LIKE PROTEIN LigA AS A PROMISING MARKER FOR SEROLOGICAL TESTING OF PATIENTS WITH LEPTOSPIROSIS


Cite item

Full Text

Abstract

Aim. To clone the DNA fragment encodi ng conservative domain of LigA protein of Leptospira interrogans into Escherichia coli and to investigate antigenic properties of constructed chimeric protein. Materials and methods. E . coli strain M15 [pREP4], recombinant plasmid pTT10 encoding cellulose-binding domain (CBD), restriction endonucleases BamHI, BglI, BglII, XbaI, T4 DNA-ligase, RNAse were used in the study. Molecular cloning of ligA gene fragment was performed using standard protocols, and expression of hybrid genes—according to «Qiagen» company’s protocols. Extraction and purification of proteins were performed using original method. Results. DNA fragment encoding immunoglobulin-like domain 5 of LigA was cloned in E.coli . Effective strain-producer of chimeric domain D5-CBD consisting of the immunoglobulin-like domain 5 of LigA, Gly-Ser spacer, and cellulose-binding domain (CBD) was obtained. The high-purity D5-CBD preparation was obtained using one-stage purification on cellulose. Antigenic specificity of this chimeric protein was studied and it was shown that it could be used as a marker for the development of diagnostic ELISA kit. Conclusion. Recombinant domain of LigA in chimeric protein produced in E.coli retains antigenic properties of native LigA protein. Obtained results confirm the feasibility to use recombinant antigen D5-CBD as a marker for development of diagnostic kits on the basis of ELISA.

Full Text

ВОЗМОЖНОСТЬ ИСПОЛЬЗОВАНИЯ РЕКОМБИНАНТНЫХ ДОМЕНОВ ИММУНОГЛОБУЛИНОПОДОБНОГО БЕЛКА LigA В КАЧЕСТВЕ ПЕРСПЕКТИВНЫХ МАРКЕРОВ ДЛЯ ТЕСТИРОВАНИЯ СЫВОРОТОК КРОВИ БОЛЬНЫХ ЛЕПТОСПИРОЗАМИ
×

About the authors

N. E Sharapova

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

Z. M Galushkina

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

N. N Poletaeva

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

N. V Lavrova

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

L. V Verkhovskaya

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

V. G Lunin

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

E. M Petrov

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

A. P Samsonova

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

Yu. V Ananyina

Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia

References

  1. Лящук А.М., Лунин В.Г., Карягина А.С. и др. Иммунизация антигенами, иммобилизированными на целлюлозе. Развитие идей А.Е. Гу рвича. Жу рн. микробиол. 2006, 4: 65-68.
  2. Лунин В.Г., Шарапова Н.Е., Тихонова Т.В. и др. Изучение иммуногенных свойств рекомбинантного целлюлозосвязывающего домена Anaerocellum thermophilum in vivo. Мол. генет. микробиол. вирусол. 2009, 1: 21-27.
  3. Лящук A.M., Лунин В.Г., Карягина А.С. и др. Получение и характеристика рекомбинантного белка TUL4 из Francisella tularensis, потенциального компонента генно-инженерной субъединичной противотуляремийной вакцины. Биотехнология. 2006, 5: 23-32.
  4. Croda J., Ramos J.G., Matsunaga J. Leptospira immunoglobulin-like proteins as a serodiagnostic marker for acute leptospirosis. J. Clin. Microbiol. 2007, 45: 1528–1534.
  5. Guerreiro H., Croda J., Flannery B. et al. Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans. Infect. Immun. 2001, 69: 4958-4968.
  6. Haake D.A., Matsunaga J. Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins. Ibid. 2002, 70: 4936–4945.
  7. Karsten H., Joanne C. The QIAexpressionist. DIAGEN GmbH, QIAGEN Inc., 1992.
  8. Koizumi N., Watanabe H. Leptospiral immunoglobulin-like proteins elicit protective immunity. Vaccine. 2004, 22: 1545-1552.
  9. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685.
  10. Matsunaga J., Barocchi M.A., Croda J. et al. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol. Microbiol. 2003, 49: 929-945.
  11. Palaniappan R.U., Chang Y.F., Jusuf S.S. et al. Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans. Infect. Immun. 2002, 70: 5924-5930.
  12. Palaniappan R.U., McDonough S.P., Divers T.J. et al. Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against leptospira interrogans serovar pomona infection. Ibid. 2006, 74: 1745-1750.
  13. Srimanote P., Wongdeethai N., Jieanampunkul P. et al. Recombinant ligA for leptospirosis diagnosis and ligA among the Leptospira spp. clinical isolates. J. Microbiol. Meth. 2008, 72: 73-81.
  14. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. 1979, 76: 4350-4354.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download

Copyright (c) 2010 Sharapova N.E., Galushkina Z.M., Poletaeva N.N., Lavrova N.V., Verkhovskaya L.V., Lunin V.G., Petrov E.M., Samsonova A.P., Ananyina Y.V.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: ПИ № ФС77-75442 от 01.04.2019 г.


This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies