Cargando…

Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitocho...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhou, Tiannan, Wei, Huixian, Wang, Jinjun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9955595/
https://www.ncbi.nlm.nih.gov/pubmed/36827343
http://dx.doi.org/10.1371/journal.pone.0279431
_version_ 1784894385453793280
author Zhou, Tiannan
Wei, Huixian
Wang, Jinjun
author_facet Zhou, Tiannan
Wei, Huixian
Wang, Jinjun
author_sort Zhou, Tiannan
collection PubMed
description Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitochondrial antibodies (AMAs) are an important diagnostic basis, especially the M2 subtype (AMA-M2) with almost 100% specificity. We selected the BCOADC-E2 protein, a mitochondrial autoantigen that reacts specifically with AMA-M2 antibodies, and carried out DNA recombination and protein mutation experiments by cloning in vitro the homologous target gene sequence BCKD that expresses the antigenic epitope of BCOADC-E2 protein, to provide experience for later exploring the effect of mutations of amino acids around the lysine in the active center of BCOADC-E2 protein on its specific binding to AMA-M2, and to lay the foundation for determining the key amino acids of BCOADC-E2 for the diagnosis and treatment of PBC. In addition, we apply this scientific research content to graduate course teaching. Experimental technology of microbial molecular ecology is a course with the cross-integration of multidisciplinary knowledge and experimental skills offered at our college since 2018. This article derives from the part of this course on the construction of recombinant plasmids. The students first constructed the recombinant plasmid pGEX-BCKD using the vector plasmid pGEX-4T1 and the target gene fragment BCKD provided by the laboratory and used this as a template to construct the pGEX-BCKD-E4A point mutation plasmid by the overlap extension PCR (SOE PCR) technique to achieve the effect of mutating the fifth amino acid glutamate in front of lysine, the active centre of the BCOADC-E2 lipid acyl binding domain, to alanine for subsequent studies. Through the research experiment, combining theoretical knowledge and experimental operation, we aim to deepen the student’s understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students’ interest in professional knowledge learning, and cultivate students’ scientific thinking and innovation consciousness. We examined the quality of the teaching through the process and summative evaluation of the students. In this study, the students successfully completed the construction of pGEX-BCKD-E4A point mutant plasmid, and the average test score increased from 40.4% before teaching to 91.1%. The teaching effect was remarkable. This kind of research experimental teaching mode has good application prospects, and other education and teachers can refer to and reference it.
format Online
Article
Text
id pubmed-9955595
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-99555952023-02-25 Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid Zhou, Tiannan Wei, Huixian Wang, Jinjun PLoS One Research Article Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitochondrial antibodies (AMAs) are an important diagnostic basis, especially the M2 subtype (AMA-M2) with almost 100% specificity. We selected the BCOADC-E2 protein, a mitochondrial autoantigen that reacts specifically with AMA-M2 antibodies, and carried out DNA recombination and protein mutation experiments by cloning in vitro the homologous target gene sequence BCKD that expresses the antigenic epitope of BCOADC-E2 protein, to provide experience for later exploring the effect of mutations of amino acids around the lysine in the active center of BCOADC-E2 protein on its specific binding to AMA-M2, and to lay the foundation for determining the key amino acids of BCOADC-E2 for the diagnosis and treatment of PBC. In addition, we apply this scientific research content to graduate course teaching. Experimental technology of microbial molecular ecology is a course with the cross-integration of multidisciplinary knowledge and experimental skills offered at our college since 2018. This article derives from the part of this course on the construction of recombinant plasmids. The students first constructed the recombinant plasmid pGEX-BCKD using the vector plasmid pGEX-4T1 and the target gene fragment BCKD provided by the laboratory and used this as a template to construct the pGEX-BCKD-E4A point mutation plasmid by the overlap extension PCR (SOE PCR) technique to achieve the effect of mutating the fifth amino acid glutamate in front of lysine, the active centre of the BCOADC-E2 lipid acyl binding domain, to alanine for subsequent studies. Through the research experiment, combining theoretical knowledge and experimental operation, we aim to deepen the student’s understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students’ interest in professional knowledge learning, and cultivate students’ scientific thinking and innovation consciousness. We examined the quality of the teaching through the process and summative evaluation of the students. In this study, the students successfully completed the construction of pGEX-BCKD-E4A point mutant plasmid, and the average test score increased from 40.4% before teaching to 91.1%. The teaching effect was remarkable. This kind of research experimental teaching mode has good application prospects, and other education and teachers can refer to and reference it. Public Library of Science 2023-02-24 /pmc/articles/PMC9955595/ /pubmed/36827343 http://dx.doi.org/10.1371/journal.pone.0279431 Text en © 2023 Zhou et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Zhou, Tiannan
Wei, Huixian
Wang, Jinjun
Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid
title Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid
title_full Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid
title_fullStr Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid
title_full_unstemmed Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid
title_short Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid
title_sort research experimental design for the construction and identification of the pgex-bckd-e4a recombinant point-mutant plasmid
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9955595/
https://www.ncbi.nlm.nih.gov/pubmed/36827343
http://dx.doi.org/10.1371/journal.pone.0279431
work_keys_str_mv AT zhoutiannan researchexperimentaldesignfortheconstructionandidentificationofthepgexbckde4arecombinantpointmutantplasmid
AT weihuixian researchexperimentaldesignfortheconstructionandidentificationofthepgexbckde4arecombinantpointmutantplasmid
AT wangjinjun researchexperimentaldesignfortheconstructionandidentificationofthepgexbckde4arecombinantpointmutantplasmid