Comparison between the two digestion methods.

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.

原发性胆汁性肝硬化（Primary biliary cirrhosis, PBC）是一种器官特异性自身免疫性疾病，最终可进展为肝硬化甚至肝癌。近年来其发病率呈上升趋势，早期诊断与治疗对PBC至关重要。在PBC的早期诊断手段中，抗线粒体抗体（anti-mitochondrial antibodies, AMAs）是重要的诊断依据，其中M2亚型（AMA-M2）的特异性几乎可达100%。本研究选取可与AMA-M2抗体特异性结合的线粒体自身抗原BCOADC-E2蛋白，通过体外克隆表达该蛋白抗原表位的同源靶基因序列BCKD，开展DNA重组与蛋白质突变实验，以期为后续探究BCOADC-E2蛋白活性中心赖氨酸周围氨基酸突变对其与AMA-M2特异性结合的影响提供实验基础，同时为明确BCOADC-E2蛋白用于PBC诊断与治疗的关键氨基酸位点奠定基础。此外，本研究将该科研内容融入研究生课程教学。《微生物分子生态学实验技术》是我院自2018年起开设的融合多学科知识与实验技能的交叉整合课程，本文内容源自该课程中重组质粒构建模块的教学实践。学生以实验室提供的载体质粒pGEX-4T1与靶基因片段BCKD为材料，首先构建重组质粒pGEX-BCKD；再以此为模板，通过重叠延伸PCR（overlap extension PCR, SOE PCR）技术构建pGEX-BCKD-E4A点突变质粒，实现将BCOADC-E2脂酰结合域活性中心赖氨酸前第5位谷氨酸突变为丙氨酸的实验目标，以供后续研究使用。通过本次研究实验，结合理论知识与实验操作，本课程旨在加深学生对DNA重组技术的理解，让学生体会实验技术的实际应用前景，激发学生对专业知识学习的兴趣，同时培养学生的科学思维与创新意识。本研究通过对学生的过程性评价与终结性评价检验教学质量。本次教学中，学生顺利完成了pGEX-BCKD-E4A点突变质粒的构建，课程测试平均得分从教学前的40.4%提升至91.1%，教学效果显著。此类研究型实验教学模式具备良好的应用前景，可供其他教育工作者参考借鉴。