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目的定点突变是通过聚合酶链式反应(PCR)等方法向目的 DNA片段中引入包括碱基的添加、删除、点突变等所需变化。体外定点突变技术是研究蛋白质结构和功能之间的复杂关系的有力工具,也是实验室中改造/优化基因常用的手段。连接蛋白43(connexin43,Cx43)是心脏表达最丰富的连接蛋白,主要分布在心室,其构成的缝隙连接(gapjunction,GJ)在心肌细胞中的电偶联中起着非常重要的作用。近年来的研究表明干细胞移植治疗心肌梗死时,细胞间电偶联障碍是导致心律失常副作用的一个重要原因。己知Cx43的磷酸化状态可以调节通道的功能,磷酸化常见的是368位点的丝氨酸磷酸化。我们对缝隙连接蛋白43基因(Cx43)的特定碱基进行定点改变,从而改变对应的氨基酸序列,并对表达产物进行研究,以利于将来探讨骨髓间充质干细胞(BMSCs)中转染突变的Cx43对其向心肌分化能力的影响,及其磷酸化状态是否对心律失常有调控作用。方法设计引物,sense:5’-CTTCAAGCAGAGCCAGCAGTCGTGCCAGCA-3’,antisense:5’TGCTGGCACGACGACTGCTGGCTCTGCTTGAAG-3’。以已构建的Pcdh-Cx43为模板,使用Stratagene基因定点突变试剂盒进行PCR扩增,用DpnI酶识别甲基化位点并将其酶切,PCR产物转化后筛选阳性克隆,提取质粒测序验证。在293FT中与其它3个穿梭质粒一起包装,再将慢病毒颗粒转染BMSCs,用嘌呤霉素筛选出阳性克隆BMSCs-mCx43,在压力培养基下传代培养。用流式细胞仪检测BMSCs-mCx43的干细胞相关表面标记表达情况,同时用western blot检测BMSCs-mCx43的蛋白表达情况。结果测序结果显示突变位点正确,荧光照片显示90%的293FT表达绿色荧光。50%的BMSCs有绿色荧光蛋白的表达。流式细胞仪检测示BMSCs-mCx43细胞CD29阳性表达94%±0.8%,CD73阳性表达92%±0.7%,几乎不表达CD34和CD105,与未转染病毒的BMSCs和转染空病毒载体的BMSCs的表面标记一致。Western blot结果显示BMSCs-mCx43中的mCx43表达强于未转染病毒的BMSCs和转染空病毒载体的BM-SCs。结论本实验研究表明使用定点突变法能成功将Cx43的368位点的丝氨酸突变成丙氨酸。用慢病毒载体转染BMSCs,能使BMSCs稳定持久表达mCx43蛋白,且转染慢病毒,不改变BMSCs的分子表面标记,是对BMSCs进行基因修饰的一种有效方法。
The purpose of site-directed mutagenesis is to introduce changes required for addition, deletion, point mutation, etc. into the DNA fragment of interest, including polymerase chain reaction (PCR) and the like. Site-directed mutagenesis in vitro is a powerful tool for studying the complex relationships between protein structure and function and is a common tool for transforming / optimizing genes in laboratories. Connexin43 (Cx43) is the most abundantly expressed connexin in the heart, mainly located in the ventricle. Gap junction (GJ) plays an important role in the galvanic coupling of cardiomyocytes. In recent years, studies have shown that stem cell transplantation in the treatment of myocardial infarction, cell-cell coupling disorder is an important cause of arrhythmic side effects. It is known that the phosphorylation state of Cx43 can regulate the function of the channel. Phosphorylation is common at the 368-site serine phosphorylation. We performed site-directed changes on specific bases of connexin 43 (Cx43) gene in order to change the corresponding amino acid sequences and to study the expression of Cx43 gene in order to facilitate the future study of Cx43 transfection in bone marrow mesenchymal stem cells (BMSCs) Its ability to differentiate into cardiomyocytes and whether its phosphorylation regulates arrhythmia. Methods Design primer, sense: 5’-CTTCAAGCAGAGCCAGCAGTCGTGCCAGCA-3 ’, antisense: 5’TGCTGGCACGACGACTGCTGGCTCTGCTTGAAG-3’. The constructed Pcdh-Cx43 was used as a template to carry out PCR amplification using the Stratagene site-directed mutagenesis kit. The methylation site was identified by DpnI enzyme digestion and the PCR product was transformed into positive clones. The plasmid was extracted and verified by sequencing. In 293FT with three other shuttle plasmid packaging, and then lentiviral particles transfected BMSCs, with puromycin screening positive clones BMSCs-mCx43, subculture under pressure medium. Flow cytometry was used to detect the expression of stem cell-associated surface markers of BMSCs-mCx43, and western blot was used to detect the protein expression of BMSCs-mCx43. Results Sequencing results showed that the mutation site was correct. Fluorescence photographs showed that 90% of 293FT expressed green fluorescence. 50% of BMSCs have green fluorescent protein expression. Flow cytometry showed that CD29 positive expression was 94% ± 0.8% and CD73 positive expression was 92% ± 0.7% in BMSCs-mCx43 cells. CD34 and CD105 were almost not expressed in BMSCs-mCx43 cells. BMSCs transfected with BMSCs and empty vector transfected BMSCs The same surface markers. Western blot results showed that the expression of mCx43 in BMSCs-mCx43 was stronger than that of non-transfected BMSCs and empty vector-transfected BM-SCs. Conclusion Our experimental study showed that the point mutation at serine 368 of Cx43 could be successfully mutated to alanine using site-directed mutagenesis. Transfection of BMSCs with lentiviral vector can make stable and long-lasting expression of mCx43 protein in BMSCs. Transfection of lentivirus without changing the molecular surface markers of BMSCs is an effective method to genetically modify BMSCs.