南京地区犬多耐药基因（MDR1）碱基缺失的PCR检测
时间:2018-08-26 21:48 来源:毕业论文 作者:毕业论文 点击:次
| 摘要： 为了研究南京地区犬多耐药基因（MDR1）的碱基缺失情况及其频率，本研究从农业大学动物医院随机抽取229只犬的血液或口腔拭子样本，采用蛋白酶K消化苯酚抽提的方法提取犬基因组DNA，根据MDR1基因设计引物，PCR扩增MDR1基因片段，然后对PCR产物进行12%中性聚丙烯酰胺凝胶电泳，硝酸银染色后对比条带的差异，对有差异的条带进行切胶、测序。结果发现229只犬中有5只苏格兰牧羊犬和1只喜乐蒂牧羊犬出现MDR1基因4 bp的碱基缺失，且均为杂合型缺失，缺失序列为TATC，且该喜乐蒂牧羊犬还发现有3个位点的碱基突变。该结果显示在南京地区苏格兰牧羊犬和喜乐蒂牧羊犬存在犬MDR1基因的碱基缺失，缺失率在该两种犬中分别为55.56%和25%。该结果为兽医临床用药和优化育种提供技术支持。27468 毕业论文关键词： MDR1；P-糖蛋白；柯利犬 The PCR detection of the canine Multidrug Resistance(MDR1) gene base deletion in Nanjing area Abstract：In order to study the base deletion and frequency of the canine Multidrug Resistance (MDR1) gene in Nanjing area, the canine genomic DNA was extracted from the blood or oral swab samples of 229 dogs randomly from the Animal Hospital of Nanjing Agricultural University by the method of proteinase K digestion and phenol extraction. According to the MDR1 gene design primers, PCR amplification of MDR1 gene fragment. The PCR products were subjected to 12% neutral polyacrylamide gel electrophoresis followed by silver nitrate staining to compare the difference in the strip. The samples of different bands were subjected to cutting and sequencing. The results showed that five Scottish Shepherd and one Shetland Sheepdog were found to have 4 bp base deletion of MDR1 of 229 dogs and were heterozygous deletions, missing the sequence TATC, and there were 3 point mutations in the Shetland Sheepdog. The results suggest that the presence of the base deletion of the MDR1 gene in the Scottish Shepherd and the Shetland Sheepdog in the Nanjing area and the base deletion frequency in the two dogs were 55.56% and 25% respectively. The results provide technical support for veterinarians in clinical application and optimization of breeding. Key words: MDR1; P-glycoprotein; collie 目 录 摘要1 关键词1 Abstract1 Key words1 引言1 1材料与方法2 1.1实验材料 2 1.1.1样品采集2 1.1.2主要试剂2 1.1.3主要仪器设备2 1.1.4引物的设计与合成2 1.2实验方法2 1.2.1犬基因组DNA提取2 1.2.2MDR1基因PCR扩增3 1.2.3PCR产物中性聚丙烯酰胺凝胶电泳3 1.2.4中性聚丙烯酰胺凝胶银染4 1.2.5 PCR产物纯化4 1.2.6 PCR产物测序4 2 结果与分析5 2.1采样结果统计5 2.2样本采集方法对PCR检测结果的影响5 2.3 MDR1突变统计与分析5 2.4 DNA测序结果统计与分析6 3讨论 8 致谢10 参考文献11 南京地区犬多耐药基因（MDR1）碱基缺失的PCR检测 引言 多耐药基因（Multidrug Resistance，MDR1）是20世纪80年代中期从中国仓鼠多药耐药细胞系基因组中克隆出的1种与药物耐受形成有关的基因序列[2]，该基因编码1280个氨基酸，经糖基化后形成170ku的P-糖蛋白[3]。P-糖蛋白的生理功能为将细胞内的毒性代谢产物泵出细胞，对组织细胞起到保护作用，在脑组织中，它能将内皮细胞中的药物排到毛细血管腔，降低脑内P-糖蛋白基质药物的浓度，从而保护脑组织[7]。在兽医临床治疗过程中经常会发现犬出现耐药现象或是对药物敏感甚至引起药物中毒甚至死亡的现象，特别是柯利犬对低剂量的伊文菌素敏感，导致犬中度死亡的病例常有发生。这与犬MDR1基因突变及P-糖蛋白异常表达有关。柯利犬常常携带MDR1基因突变，导致P-糖蛋白截断表达，使P-糖蛋白功能缺失，药物无法被转运到细胞外，导致动物中度甚至死亡。 (责任编辑：qin) |