人SNCA基因真核表达载体的构建及其在PC12细胞中的表达

1、人SNCA基因真核表达载体的构建及其在PC12细胞中的表达         11-01-31 15:53:00     编辑：studa20                作者：钱进军, 程言博, 刘春风, 杨亚萍, 刘康永 【摘要】  目的: 构建含人野生型及致病突变A30P、 A53T SNCA基因的重组

2、真核表达载体pEGFPC3SNCA, 并通过稳定转染获得过表达人野生型及致病突变A30P、 A53T synuclein(SNCA)的单克隆PC12细胞株。方法: 通过RTPCR方法扩增SNCA基因, TA克隆后测序。在此基础上利用单核苷酸差异引物定点诱变法相继构建含SNCA基因编码区EcoR I、 BamH I两酶切位点的同义突变及其致病突变G88C（Ala30 Pro）、 G209A（Ala53Thr）的重组真核表达载体pEGFPC3SNCA, 并以PCR、 双酶切、 测序鉴定。以重组质粒pEGFPC3SNCA通过脂质体转染方法转染PC12细胞; 以G418进行筛选; 以有限稀释法进行亚克

3、隆获得稳定过表达人野生型及致病突变A30P、 A53T synuclein的单克隆PC12细胞株, 并以稳定转染pEGFPC3的PC12细胞作为对照组细胞, 通过RTPCR、 Western blot及荧光显微镜鉴定各单克隆PC12细胞株。结果: PCR、 酶切及测序证明重组真核表达载体pEGFPC3SNCA构建成功。RTPCR、 Western blot及荧光显微镜确认目的基因序列在PC12细胞稳定过表达。结论: 成功地构建SNCA基因WT及A30P与A53T突变型的重组真核表达载体pEGFPC3SNCA; 成功获得过表达人野生型及致病突变A30P、 A53T synuclein的单克隆PC

4、12细胞株。 【关键词】  synuclein（SNCA） 致病突变 真核表达载体pEGFP C3 稳定转染 PC12细胞Abstract  AIM: To construct recombinant eukaryotic expression vectors pEGFPC3SNCA containing human wildtype (WT) and  pathogenic mutations A30P, A53T synuclein (SNCA), and to obtain monoclonal PC12 cell lines overexpressing

5、human wildtype and pathogenic mutations A30P, A53T synuclein by stable transfection. METHODS: Human wild type SNCA gene was cloned by using RTPCR. By TA extension cloning, the gene was ligated with Tvector and sequenced. Based on it, the recombinant eukaryotic expression vectors containing wild type

6、 SNCA synonymous mutation or its G88C (Ala30Pro) and G209A (Ala53Thr) pathogenic mutation were constructed by sitedirected mutagensis using primer variance in mononucleotide. And different recombinant plasmids pEGFPC3SNCA were identified with PCR, restriction enzyme digestion and DNA sequencing. PC1

7、2 cells were transfected with different recombinant plasmids pEGFPC3SNCA by liposome transfection method, screened with G418, and subcloned by limited dilution method to obtain different monoclonal PC12 cell lines stably overexpressing human wildtype, A30P or A53T synuclein, respectively, and PC12 c

8、ells stably transfected with pEGFPC3 were used as control group. These monoclonal PC12 cell lines were identified with RTPCR, Western blot and fluorescence microscopy. RESULTS: According to result of PCR, the double digestion and gene sequencing, it was comfirmed that recombinant eukaryotic expressi

9、on vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutation had been constructed successfully. By means of RTPCR, Western blot and fluorescence microscope, it was comfirmed that objective gene sequences in PC12 cells were stably overexpressed. CONCLUSION

10、: The recombinant pEGFPC3SNCA vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutations had been constructed successfully. The transfected monoclonal PC12 cells are obtained in which three SNCA gene isoforms had stable expression.Keywordssynuclein（SNCA）;

11、 pathogenic mutation; eukaryotic expression vector pEGFPC3; stable transfection; PC12 cell多巴胺能神经元中出现路易小体（Lewy body, LB）是帕金森病（Pakinsons disease, PD）的特征性病理改变, SNCA基因编码的synuclein在病理状态下聚集形成不溶性纤维, 构成了LB的主要成分, 而Ala30Pro、 Ala53Thr的致病突变与家族性PD相关1, 2。synuclein及其Ala30Pro、 Ala53Thr致病突变体在PD的病理机制中可能起着十分重要的作用, 我们通

12、过构建含绿色荧光蛋白的SNCA同义突变基因及其Ala30Pro与Ala53Thr致病突变基因的真核表达载体pEGFPC3SNCA质粒, 并以此转染PC12细胞, 建立稳定表达该基因蛋白及变异蛋白的多巴胺能PC12细胞模型。1  材料和方法1.1  材料  克隆载体pTZ57R与真核表达载体pEGFPC3购自Promega公司; E.coli   DH5菌株、 培养基RPMI1640购自Invitrogen公司; PC12细胞株购自中国科学院上海细胞所; 小牛血清购自杭州四季青公司; Taq酶购自TaKaRa公司; 总RNA提取试剂为Gibco的

13、TRIzol试剂; 反转录PCR试剂盒购自Fermanas公司; Lipofectamine2000转染试剂盒购自GbicoBRL公司; G418购自Mediatech公司; 胰蛋白酶、 兔抗人synuclein mAb购自Sigma公司; HRP标记的羊抗兔IgG的二抗购自DAKO公司; ECL显色剂购自Amersham公司。1.2  方法1.2.1  PCR引物的合成  根据GenBank中相应cDNA基因序列, 自行设计引物, 委托上海英骏生物有限公司合成, 引物序列: P1: 5GTGTGGTGTAAAGGAATTCATT3; P2: 5CGCGGATCC

14、AGAAACTGGGAGCAAAGATA3。1.2.2  总RNA的提取及RTPCR扩增  在50 L反应体系中加入10 ×buffer 5L, 10 mmol/L, dNTP 1 L, 引物1、 2 各1 L, 5 U/L Taq酶0.5 L, 模板cDNA 2 L, ddH2O 40 L, 计反应体系50 L。PCR反应条件为94 5 min, 94 30 s, 55  30 s, 72 45 s, 后3步反复进行30个循环。扩增产物用Uuiq10柱式DNA胶回收试剂盒提纯。1.2.3  野生型SNCA基因的TA克隆  取胶回收产

15、物7.5 L加入1 L T vector、 1 L 10×ligation buffer、 0.5 L T4 DNAligase, 混合为10 L连接反应体系于4过夜。将10 L连接液与200 L感受态细菌混匀, 冰浴30 min后于42水浴90 s, 置冰浴2 min。加入600 L不含氨苄青霉素的LB培养液, 37、 225 r/min摇床45 min, 将细菌涂布于氨苄青霉素平板上, 37倒置过夜。取单克隆菌落加入含氨苄青霉素的LB(100mg/L) 37、 225r/min摇床8h, 用Uuiq10柱式质粒小抽提试剂盒抽取质粒, 作PCR及双酶切鉴定, 阳性者送检测序。1.2

16、.4  同义突变SNCA及Ala30Pro、 Ala53Thr致病突变SNCA的构建在野生型SNCA基因编码区内部存在EcoRI、 BamHI两酶切位点, 以野生型SNCA基因的TA克隆质粒为模板, 利用单核苷酸差异引物定点诱变法针对两酶切位点进行同义突变,  在此基础上同法构建G88C（Ala30Pro）、 G209A（Ala53Thr）已知致病突变体, 并对SNCA同义突变及Ala30Pro、 Ala53Thr致病突变基因进行TA克隆。1.2.5  含同义突变及致病突变Ala30Pro、 Ala53Thr的SNCA基因重组真核表达载体pEGFPC3SNCA的构

17、建  分别以SNCA同义突变及Ala30Pro、 Ala53Thr致病突变基因的TA克隆质粒为模板, 以P1、 P2引物, 以高保真Taq酶PCR扩增3种不同SNCA基因片段, 扩增条件同前。分别取相应提纯的PCR产物及pEGFPC3质粒5L, 加入BamHI0.5L、 EcoR I0.5 L、 10×buffer2 L、 ddH2O12 L, 构成20 L反应体系37 酶切5 h。将3种不同SNCA的PCR双酶切产物电泳胶回收后分别与pEGFPC3空载体双酶切胶回收产物混合进行连接, 转化细菌后用Uuiq10柱式质粒小量抽提试剂盒抽取质粒, 作PCR扩增、 酶切及测序鉴定

18、。1.2.6  人同义突变（野生型）及致病突变Ala30Pro、 Ala53Thr SNCA基因在PC12细胞中的稳定过表达  PC12细胞长至80%融合后, 进行转染实验3。取2 g质粒溶于100 L无血清RPMI1640液, 再取3 L脂质体溶于100 L无血清RPMI1640, 静置5 min后将2液体混匀, 室温静置30 min; 培养细胞用无血清RPMI1640洗1次, 加800 L无血清RPMI1640, 滴入转染混合液, 摇匀; 置37 50 mL/L CO2的培养箱内培养524 h, 换液为RPMI1640(100 mL/L FCS), 继续培养至总转染时间

19、为4872 h。pEGFPC3SNCA质粒转染PC12细胞48 h后, 加入含500 mg/L G418的选择性培养液, 23 d更换新的选择性培养液, 约5 d后见细胞死亡, 2周后见细胞克隆, 将具有G418抗性的细胞用200 mg/L G418维持培养基通过有限稀释法将细胞稀释至约130个/mL, 转移至96孔培养板中, 每孔加100 L细胞悬液, 培养45 d后, 在显微镜下可见到小的细胞克隆, 期间更换选择性培养液, 第89天时, 肉眼可见细胞克隆。转移至24孔板继续用选择性培养液扩大培养。再用有限稀释法进行2次亚克隆。将单克隆细胞扩大培养备用, 并于荧光显微镜下观察摄片。1.2.7

20、  RTPCR检测转染PC12细胞中SNCA转录  收集筛选获得的阳性细胞, 用TRIzol抽提其总mRNA, 按TFermanas逆转录试剂盒将mRNA逆转录成cDNA, 取5 L cDNA为模板, 用SNCA特异性引物扩增SNCA基因, 同时用转pEGFPC3的PC12细胞作阴性对照。1.2.8  Western blot检测稳定转染的PC12pEGFPC3SNCA单克隆细胞synuclein的表达  将筛选获得的含同义突变及A30P、 A53T致病突变SNCA基因的PC12pEGFPC3SNCA细胞单克隆分别胰酶消化, 用含200 g/L G418的培养基37 50 mL/L CO2培养3 d后消化收集细胞, PBS洗涤后加入蛋白裂解液超声裂解, 4、 12000 r/min离心20 min,  取上清分装, 取适量稀释后进行蛋白定量, 部分存于-80待用。用上样缓冲液将各样品蛋白