第四节载脂蛋白CⅢ基因型检测

第四节　载脂蛋白CⅢ基因型检测

　　载脂蛋白CⅢ基因位于11号染色体与载脂蛋白AⅠ-AⅣ形成一基因族，它位于载脂蛋白AⅠ下游，有三个内含子和四个外显子。对载脂蛋白CⅢ基因3'末端非编码区C-G对换产生一个Sac-Ⅰ酶切位点与冠心病的关系未有定论。用多聚酶链反应（PCR）扩增载脂蛋白CⅢ基因3'末端233bp，再用Sac-1酶切扩增产物，可以检测载脂蛋白CⅢ基因3'末端的突变及其与动脉粥样硬化相关性。本节采用PCR-RFLP检测ApoCⅢ基因型。

　　一、仪器与试剂

　　1．仪器：电泳仪，基因扩增仪。

　　2．试剂：TaqDNA聚合酶，dNTP，DNA参考物，Sca-1酶。

　　二、操作方法

　　1.模DNA提取

　　取用EDTA抗凝全血100μl加试剂(0.32mol/L蔗糖，5mmol/l MgCl₂,1% TritonX-100,0.01mol/L Tris-HClpH7.6)，振摇至溶解均匀透明，3000r/min离心10min,弃上清，沉淀部分加0.9NaCl溶液洗一次，加预冷试剂Ⅱ(75mmol/l NaCl,24mmol/L EDTA)100μl，20%SDS15μl，蛋白酶K10μl（6g/L）于65℃水浴4h，加200μl酚和氯仿：异戊醇（24：1）各提两次，上清加无水乙醇1.0ml放置于-20℃2h，以12kr/min离心5min，真空干燥后加50μlTE缓冲液溶解，4℃保存备用。

　　2．多聚酶链反应

　　Primer1：5'-GTG ACC GAT GGC TTC AGT TCC CTG-3'，primer2:5'-GGt AGG AGA GCA CTC AGA ATA CA-3'。反应总体积为50μl，扩增缓冲液所含物质终浓度为：Tris-HCl，pH8.3，67mmol/L；MgCl₂25mmol/L；KCl 50mmol/L；明胶0.01%，dNTP各0.2～0.5μmol/L，模板约10μg，加40μl液体石蜡，94℃预变性4min，加Tag聚合酶1.5U，以93℃1min、65℃1.5min、72℃1.5min，循环30次。

　　3．扩增产物酶切

　　取扩增产物20μl加入NH₄AC20μl，再加300μl无水乙醇混均置-20℃1h，10000r/min离心7min弃上清，干燥后加Sac-1酶液10μl37℃保温2h。

　　4.扩增产物碱基对分子量测定

　　采用GB公司产DNa marker,以已知DNA片段bp数相对应的电泳迁移距离，取半对数作图制成标准曲线，再根据等测定扩增产物DNA片段移动距离在标准曲线上求出其bp数。

　　三、结果分析

　　采用本节设计引物扩增产物为ApoCⅢ基因3'非编码区233bp，该产物经Sac-1酶切可出现三种结果；①酶切后仍为一条带，电泳迁移率未发生改变，为S₁S₁纯合子；②酶切后除原始带外又增加了两条带，查标准DNa marker曲线在158bp和75bp相同，为S₂S₂杂合子；③酶切后原始带消失，出现两条新带，其电泳迁移率与158bp和75bp相同，为S₂S₂纯合子。S₂为扩增的基因产物中C-G对换增加一个酶切位点。

　　检查100名正常人中有S₂杂合子28名，纯合子2名；68名冠心病人中S₂杂合16名，纯合子4名，它们的频率变化见表（19-4）。S₂基因频率两组间无明显差别。

表19-4 载脂蛋白CⅢ基因Sac-Ⅰ酶切频率表

　　 X=0.079,p＞0.05(两组比较)

（徐琼）

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