PCSK9基因D374Y突变体转基因猪的制备与分析
发布时间：2018-08-03 21:53
【摘要】：【目的】前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/Kexin type 9,PCSK9)基因是人类高胆固醇血症(autosomal dominant hypercholesterolemia,ADH)的主效基因之一,其获得型突变与人类家族性高胆固醇血症有直接的关系。PCSK9-D374Y突变体对低密度脂蛋白受体(low density lipoprotein receptor,LDLR)的降解能力比野生型蛋白强十倍,增加了患高胆固醇血症的风险,从而加速动脉粥样硬化的进程。猪心血管系统和血脂代谢方面与人类非常相近,成为研究动脉粥样硬化疾病的理想模型之一。然而自然发病的猪缺乏,且诱导病征发生缓慢。因此拟利用体细胞克隆技术制备PCSK9获得型突变体转基因猪,以模拟动脉血管的病理学变化,加速发病进程,为动脉粥样硬化的研究提供理想的动物模型。【方法】研究使用人PCSK9基因D374Y突变体载体,用电转染的方法将其整合到五指山小型猪近交系胎儿成纤维细胞中,并通过体细胞核移植技术获得了人PCSK9基因D374Y突变体转基因猪个体。通过Southern-blot、实时荧光定量PCR、Western-blot等方法,分别从DNA、RNA、蛋白的水平检测了人PCSK9基因在转基因猪肝脏中的整合表达情况。同时,通过组织化学染色与H.E.染色的方法对转基因猪进行了组织学检测。【结果】转基因阳性细胞集落在药筛的第3天开始出现,至第7天形成较大的单克隆点,且PCR检测结果显示扩增产物可以拼接为完整片段,说明外源片段在基因组中具有完整性;将筛选得到的阳性细胞作为体细胞克隆的供体细胞,通过体细胞核移植技术获得了转基因猪个体。PCR及Southern-blot检测结果显示,D374Y-PCSK9基因可以完整的插入猪的基因组中,且有串联重复现象;RT-PCR和QPCR检测结果表明,人PCSK9基因能在猪肝脏内正常转录且不影响猪内源性PCSK9基因的转录,且在其它内脏器官,如心、脾、肺、肾也能检测人PCSK9基因的表达,而猪内源性PCSK9基因在这些组织中表达量很低;Western-blot检测结果与RNA水平的检测类似。这些结果说明人D374Y-PCSK9基因成功整合到猪基因猪中,且能够正常转录与翻译。通过组织化学染色发现,与野生型猪肝脏相比,克隆猪肝脏中LDLR蛋白水平极显著低于野生型。另外,对克隆猪进行H.E.染色后发现其肝脏组织有明显的病理学变化,该结果说明,LDLR水平的急剧下降有可能是导致肝脏病变的原因。【结论】成功获得了人PCSK9基因D374Y突变体的克隆猪;与野生型猪肝脏相比,克隆猪肝脏中LDLR水平显著降低,并且克隆猪肝脏发生了明显病变。
[Abstract]:[objective] the proprotein invertase lysogenin 9 (proprotein convertase subtilisin/Kexin type 9 / PCSK9 gene is one of the major genes of (autosomal dominant hypercholesterolemia. Its acquired mutation was directly related to human familial hypercholesterolemia. PCSK9-D374Y mutant was 10 times more capable of degrading low density lipoprotein receptor (low density lipoprotein receptor LDLR than wild-type protein, which increased the risk of hypercholesterolemia. Thus speeding up the process of atherosclerosis. The cardiovascular system and lipid metabolism of pigs are very similar to those of human beings, and become one of the ideal models for the study of atherosclerotic diseases. However, naturally occurring pigs are deficient and slow to induce symptoms. Therefore, we intend to use somatic cell cloning technique to prepare PCSK9 acquired mutant transgenic pigs in order to mimic the pathological changes of arterial vessels and accelerate the pathogenesis of the disease. To provide an ideal animal model for the study of atherosclerosis. [methods] the D374Y mutant vector of human PCSK9 gene was used to integrate the D374Y mutant vector into fetal fibroblasts of Wuzhishan mini-pig inbred line by electrotransfection. Human PCSK9 gene D374Y mutant transgenic pig was obtained by somatic cell nuclear transfer technique. The integrative expression of human PCSK9 gene in transgenic porcine liver was detected by Southern-blot and real-time fluorescence quantitative PCR Western-blot. At the same time, by histochemical staining and H. E. The staining method was used to detect the histology of transgenic pigs. [results] the colony of transgenic positive cells began to appear on the third day of screening, and formed a large monoclonal spot on the 7th day. The results of PCR showed that the amplified products could be spliced into complete fragments, indicating that the foreign fragments had integrity in the genome, and the selected positive cells were taken as donor cells cloned from somatic cells. By means of somatic cell nuclear transfer technique, the results of .PCR and Southern-blot analysis showed that the gene of D374Y-PCSK9 could be inserted into the genome of pig completely, and there were tandem repeats in the genome. The results of RT-PCR and QPCR analysis showed that D374Y-PCSK9 gene could be inserted into pig genome completely. Human PCSK9 gene can be transcribed normally in pig liver without affecting the transcription of porcine endogenous PCSK9 gene, and the expression of human PCSK9 gene can also be detected in other visceral organs, such as heart, spleen, lung and kidney. However, the expression of porcine endogenous PCSK9 gene in these tissues was very low. The result of Western-blot analysis was similar to that of RNA level. These results suggest that human D374Y-PCSK9 gene is successfully integrated into porcine pigs and can be transcribed and translated normally. Histochemical staining showed that the level of LDLR protein in cloned pig liver was significantly lower than that in wild type pig liver. In addition, H. E. The pathological changes of liver tissue were found after staining, which indicated that the sharp decrease of LDLR level might be the cause of liver lesion. [conclusion] the cloned pig of human PCSK9 gene D374Y mutant was successfully obtained. Compared with wild-type pig liver, the LDLR level in cloned pig liver was significantly lower and the cloned pig liver had obvious pathological changes.
【作者单位】： 中国农业科学院北京畜牧兽医研究所;
【基金】：国家自然科学基金(31572378) 国家高技术研究发展计划(863计划,2012AA020603)
【分类号】：R-332;R543.5
本文编号：2163105
[Abstract]:[objective] the proprotein invertase lysogenin 9 (proprotein convertase subtilisin/Kexin type 9 / PCSK9 gene is one of the major genes of (autosomal dominant hypercholesterolemia. Its acquired mutation was directly related to human familial hypercholesterolemia. PCSK9-D374Y mutant was 10 times more capable of degrading low density lipoprotein receptor (low density lipoprotein receptor LDLR than wild-type protein, which increased the risk of hypercholesterolemia. Thus speeding up the process of atherosclerosis. The cardiovascular system and lipid metabolism of pigs are very similar to those of human beings, and become one of the ideal models for the study of atherosclerotic diseases. However, naturally occurring pigs are deficient and slow to induce symptoms. Therefore, we intend to use somatic cell cloning technique to prepare PCSK9 acquired mutant transgenic pigs in order to mimic the pathological changes of arterial vessels and accelerate the pathogenesis of the disease. To provide an ideal animal model for the study of atherosclerosis. [methods] the D374Y mutant vector of human PCSK9 gene was used to integrate the D374Y mutant vector into fetal fibroblasts of Wuzhishan mini-pig inbred line by electrotransfection. Human PCSK9 gene D374Y mutant transgenic pig was obtained by somatic cell nuclear transfer technique. The integrative expression of human PCSK9 gene in transgenic porcine liver was detected by Southern-blot and real-time fluorescence quantitative PCR Western-blot. At the same time, by histochemical staining and H. E. The staining method was used to detect the histology of transgenic pigs. [results] the colony of transgenic positive cells began to appear on the third day of screening, and formed a large monoclonal spot on the 7th day. The results of PCR showed that the amplified products could be spliced into complete fragments, indicating that the foreign fragments had integrity in the genome, and the selected positive cells were taken as donor cells cloned from somatic cells. By means of somatic cell nuclear transfer technique, the results of .PCR and Southern-blot analysis showed that the gene of D374Y-PCSK9 could be inserted into the genome of pig completely, and there were tandem repeats in the genome. The results of RT-PCR and QPCR analysis showed that D374Y-PCSK9 gene could be inserted into pig genome completely. Human PCSK9 gene can be transcribed normally in pig liver without affecting the transcription of porcine endogenous PCSK9 gene, and the expression of human PCSK9 gene can also be detected in other visceral organs, such as heart, spleen, lung and kidney. However, the expression of porcine endogenous PCSK9 gene in these tissues was very low. The result of Western-blot analysis was similar to that of RNA level. These results suggest that human D374Y-PCSK9 gene is successfully integrated into porcine pigs and can be transcribed and translated normally. Histochemical staining showed that the level of LDLR protein in cloned pig liver was significantly lower than that in wild type pig liver. In addition, H. E. The pathological changes of liver tissue were found after staining, which indicated that the sharp decrease of LDLR level might be the cause of liver lesion. [conclusion] the cloned pig of human PCSK9 gene D374Y mutant was successfully obtained. Compared with wild-type pig liver, the LDLR level in cloned pig liver was significantly lower and the cloned pig liver had obvious pathological changes.
【作者单位】： 中国农业科学院北京畜牧兽医研究所;
【基金】：国家自然科学基金(31572378) 国家高技术研究发展计划(863计划,2012AA020603)
【分类号】：R-332;R543.5
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