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[Genetic Recombiniation and Protein Expression Detection of Listeria-based tuberculosis Vaccine Candidates.]
Ren, Chen-Yan; Liu, Si-Jing; Pu, Qi-Kang; Jiang, Ming-Juan; Zhou, Meng-Ying; Wang, Chuan.
Afiliação
  • Ren CY; Department of Public Health Laboratory Science,West China School of Public Health,Sichuan University,Chengdu 610041,China.
  • Liu SJ; Department of Public Health Laboratory Science,West China School of Public Health,Sichuan University,Chengdu 610041,China.
  • Pu QK; Department of Public Health Laboratory Science,West China School of Public Health,Sichuan University,Chengdu 610041,China.
  • Jiang MJ; Department of Public Health Laboratory Science,West China School of Public Health,Sichuan University,Chengdu 610041,China.
  • Zhou MY; Department of Public Health Laboratory Science,West China School of Public Health,Sichuan University,Chengdu 610041,China.
  • Wang C; Department of Public Health Laboratory Science,West China School of Public Health,Sichuan University,Chengdu 610041,China.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 47(6): 819-824, 2016 Nov.
Article em Zh | MEDLINE | ID: mdl-28598105
ABSTRACT

OBJECTIVES:

Genetic construction of tuberculosis vaccine candidates based on Listeria(L.) monocytogenes,L.ivanovii,and evaluation their protein expression,in order to provide a novel method for research on tuberculosis controlling.

METHODS:

Two kinds of gene cassettes carrying tuberculosis antigen encoding gene Rv3875 or Rv0129c were inserted into targeting vector harboring L.monocytogenes,L.ivanovii homologous sequences via genetic connection methods and plasmid transformation technology in vitro.Targeting plasmids were electroporated into L.monocytogenes,L.ivanovii,and the recombinant strains were experienced serial passage at 42 °C and 30 °C.Subsequently,the tuberculosis antigen gene cassettes in targeting plasmids were integrated into L.monocytogenes and L.ivanovii attenuated strain (knocking out of virulence gene actA and plcB) and L.ivanovii wild type strain by homologous recombination and gene targeting technology.The recombinant strains were screened by blue-white spot and antibiotic resistance test;the intracellular and extracellular proteins of the recombinant strains were tested by Western blot.

RESULTS:

Five recombination strains carried antigen gene cassette were constructed,and the recombinant genome were confirmed by PCR and sequencing.No erythromycin resistance gene was found in 5 strains,which was coincident to expection.Recombination strains Li-Rv0129c,Li-ΔactAplcB-Rv0129c and Li-ΔactAplcB-Rv3875 expressed Mycobacterium tuberculosis antigenic protein,Ag85C or ESAT-6,as expected.But L.monocytogenes strains did not express proper antigenic protein.

CONCLUSIONS:

Three novel L.ivanovii-based tuberculosis vaccine candicates,carrying Mycobacterium tuberculosis Rv0129c antigen gene cassette (coding for Ag85C) or Rv3875 gene cassette (coding for ESAT-6),and expressing relevant antigenic proteins have been successfully selected.
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Recombinação Genética / Vacinas contra a Tuberculose / Listeria / Antígenos de Bactérias Tipo de estudo: Diagnostic_studies Idioma: Zh Revista: Sichuan Da Xue Xue Bao Yi Xue Ban Ano de publicação: 2016 Tipo de documento: Article País de afiliação: China
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Recombinação Genética / Vacinas contra a Tuberculose / Listeria / Antígenos de Bactérias Tipo de estudo: Diagnostic_studies Idioma: Zh Revista: Sichuan Da Xue Xue Bao Yi Xue Ban Ano de publicação: 2016 Tipo de documento: Article País de afiliação: China