{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,21]],"date-time":"2025-10-21T15:50:06Z","timestamp":1761061806472,"version":"3.37.3"},"reference-count":39,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2023,2,24]],"date-time":"2023-02-24T00:00:00Z","timestamp":1677196800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81873877"],"award-info":[{"award-number":["81873877"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100013151","name":"Scientific Research Foundation for Returned Scholars of Ministry of Education","doi-asserted-by":"publisher","award":["20151098"],"award-info":[{"award-number":["20151098"]}],"id":[{"id":"10.13039\/501100013151","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>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\u2019s understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students\u2019 interest in professional knowledge learning, and cultivate students\u2019 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.<\/jats:p>","DOI":"10.1371\/journal.pone.0279431","type":"journal-article","created":{"date-parts":[[2023,2,24]],"date-time":"2023-02-24T18:49:26Z","timestamp":1677264566000},"page":"e0279431","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3553-6572","authenticated-orcid":true,"given":"Tiannan","family":"Zhou","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huixian","family":"Wei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5723-1799","authenticated-orcid":true,"given":"Jinjun","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2023,2,24]]},"reference":[{"issue":"3","key":"pone.0279431.ref001","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1080\/00325481.2021.1885945","article-title":"Similar clinical outcome of AMA immunoblot-M2-negative compared to immunoblot-positive subjects over six years of follow-up","volume":"133","author":"S Zandanell","year":"2021","journal-title":"Postgraduate medicine"},{"key":"pone.0279431.ref002","doi-asserted-by":"crossref","first-page":"102328","DOI":"10.1016\/j.jaut.2019.102328","article-title":"The challenges of primary biliary cholangitis: What is new and what needs to be done","volume":"105","author":"BT Beretta-Piccoli","year":"2019","journal-title":"Journal of autoimmunity"},{"issue":"1","key":"pone.0279431.ref003","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1515\/cclm-2017-0249","article-title":"The diagnostic accuracy of biomarkers for diagnosis of primary biliary cholangitis (PBC) in anti-mitochondrial antibody (AMA)-negative PBC patients: a review of the literature","volume":"56","author":"F De Liso","year":"2018","journal-title":"Clinical Chemistry and Laboratory Medicine (CCLM)."},{"issue":"13","key":"pone.0279431.ref004","doi-asserted-by":"crossref","first-page":"1809","DOI":"10.1080\/14656566.2016.1218471","article-title":"Obeticholic acid for the treatment of primary biliary cholangitis.","volume":"17","author":"AH Ali","year":"2016","journal-title":"Expert opinion on pharmacotherapy"},{"issue":"10","key":"pone.0279431.ref005","doi-asserted-by":"crossref","first-page":"2547","DOI":"10.1007\/s10620-018-5074-8","article-title":"Are Clinicians Ready for Safe Use of Stratified Therapy in Primary Biliary Cholangitis (PBC)? 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