EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

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• 作者：Samira Seif ; Fereshteh Kazemi ; Elham Gholami…
• 关键词：Reporter gene ; EGFP ; Luciferase ; Leishmania major ; In vivo infectivity
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2016
• 出版时间：May 2016
• 年：2016
• 卷：100
• 期：9
• 页码：3923-3934
• 全文大小：719 KB
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• 作者单位：Samira Seif (1) (2)
  Fereshteh Kazemi (1) (3)
  Elham Gholami (1)
  Negar Seyed (1)
  Yasaman Taslimi (1)
  Sima Habibzadeh (1)
  Bahareh Azarian (4)
  Shahram Jamshidi (2)
  Mehrdad Hashemi (3)
  Sima Rafati (1)
  Tahereh Taheri (1)
  
  1. Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran
  2. Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
  3. Department of Genetics Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran
  4. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major EGFP or L. major EGFP-LUC) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wild-types that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study.