Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a
Abstract Background Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or a...
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Wiley
2024-09-01
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| Series: | Immunity, Inflammation and Disease |
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| Online Access: | https://doi.org/10.1002/iid3.70021 |
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| author | Liang Yu Wang Yongbo Yang Shengjun Tan Jia Xu Ya Liao Guoyang Ma Linna |
| author_facet | Liang Yu Wang Yongbo Yang Shengjun Tan Jia Xu Ya Liao Guoyang Ma Linna |
| author_sort | Liang Yu |
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| description | Abstract Background Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector. Methods In this study, the major antigen genes P1a of MP adhesion factor P1(3862‐4554 bases) and P30a of P30(49‐822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS‐P1a or NS‐P30a. The recombinant pHW2000 plasmids containing NS‐P1a or NS‐P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU‐P1a and rFLU‐P30a were rescued. RT‐PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU‐P1a and rFLU‐P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy. Results P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT‐PCR identification of the recombinant viruses rFLU‐P1a and rFLU‐P30a showed that P1a (693 bp), P30a (774 bp), NS‐P1a (1992bp) and NS‐P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins. Conclusion The recombinant viruses rFLU‐P1a and rFLU‐P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU‐P1a or rFLU‐P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human. |
| format | Article |
| id | doaj-art-e9ab426a187b4d37a20b6b7bfd7d5fc2 |
| institution | OA Journals |
| issn | 2050-4527 |
| language | English |
| publishDate | 2024-09-01 |
| publisher | Wiley |
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| series | Immunity, Inflammation and Disease |
| spelling | doaj-art-e9ab426a187b4d37a20b6b7bfd7d5fc22025-08-20T02:16:50ZengWileyImmunity, Inflammation and Disease2050-45272024-09-01129n/an/a10.1002/iid3.70021Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30aLiang Yu0Wang Yongbo1Yang Shengjun2Tan Jia3Xu Ya4Liao Guoyang5Ma Linna6Department of Clinical Laboratory The First People's Hospital of Yunnan Province Kunming ChinaDepartment of Clinical Laboratory The First People's Hospital of Yunnan Province Kunming ChinaDepartment of Clinical Laboratory The First People's Hospital of Yunnan Province Kunming ChinaDepartment of Clinical Laboratory The First People's Hospital of Yunnan Province Kunming ChinaDepartment of Clinical Laboratory The First People's Hospital of Yunnan Province Kunming ChinaThe Fifth Department of Biological Products Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College Kunming ChinaDepartment of Medical Laboratory Technique Kunming Medical University Haiyuan College Kunming ChinaAbstract Background Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector. Methods In this study, the major antigen genes P1a of MP adhesion factor P1(3862‐4554 bases) and P30a of P30(49‐822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS‐P1a or NS‐P30a. The recombinant pHW2000 plasmids containing NS‐P1a or NS‐P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU‐P1a and rFLU‐P30a were rescued. RT‐PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU‐P1a and rFLU‐P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy. Results P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT‐PCR identification of the recombinant viruses rFLU‐P1a and rFLU‐P30a showed that P1a (693 bp), P30a (774 bp), NS‐P1a (1992bp) and NS‐P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins. Conclusion The recombinant viruses rFLU‐P1a and rFLU‐P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU‐P1a or rFLU‐P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human.https://doi.org/10.1002/iid3.70021genetic engineeringinfluenza virus vectorMycoplasma pneumoniaevaccine |
| spellingShingle | Liang Yu Wang Yongbo Yang Shengjun Tan Jia Xu Ya Liao Guoyang Ma Linna Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a Immunity, Inflammation and Disease genetic engineering influenza virus vector Mycoplasma pneumoniae vaccine |
| title | Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a |
| title_full | Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a |
| title_fullStr | Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a |
| title_full_unstemmed | Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a |
| title_short | Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a |
| title_sort | research of recombinant influenza a virus as a vector for mycoplasma pneumoniae p1a and p30a |
| topic | genetic engineering influenza virus vector Mycoplasma pneumoniae vaccine |
| url | https://doi.org/10.1002/iid3.70021 |
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