CD59基因siRNA表达载体的构建及CD59短肽封条对HeLa细胞凋亡作用的研究

发布时间：2018-03-27 12:47

  本文选题：CD59　切入点：pSUPER载体　出处：《青岛大学》2008年硕士论文

【摘要】： 目的:构建针对CD59基因的pSUPER retro RNAi逆转录病毒载体,为下一步研究肿瘤细胞内CD59 RNAi奠定基础。利用本课题组设计并合成的CD59短肽封条,检测其对HeLa细胞凋亡的作用,为肿瘤的治疗提供一条新的思路。 方法:根据GenBank提供的CD59基因mRNA的序列及siRNA的设计原则设计RNA干扰序列及一条对照序列,以CD59基因mRNA序列的220-238nt的19个bp片段作为靶序列,设计并合成两端含有酶切位点的60个碱基的寡核苷酸链。寡核苷酸链退火后用T_4连接酶连接至线性化的质粒pSUPER.retro.neo+GFP中,转化大肠杆菌JM109,提取质粒。然后通过酶切,PCR,测序鉴定重组质粒。50mg/L CD59短肽封条作用于HeLa细胞24h后,用透射电镜检测HeLa细胞的凋亡情况;用MTT的方法来检测HeLa细胞增殖抑制率;利用免疫组织化学方法检测survivin,caspase-3,bax的表达水平。 结果:经酶切鉴定,PCR鉴定及测序结果表明成功地构建了CD59基因的pSUPER retroRNAi逆转录病毒载体。透射电镜结果表明,CD59短肽封条能使HeLa细胞的胞核发生碎裂。MTT实验表明转CD59基因的HeLa细胞+短肽的细胞组增殖抑制率大于HeLa细胞+短肽组(P＜0.01)。免疫组化实验显示,转CD59基因的HeLa细胞+短肽组和正常的HeLa细胞+短肽组相比较,survivin表达水平降低,差别有统计学意义(P＜0.05);而caspase-3表达水平增高,差别有统计学意义(P＜0.05);bax的表达量各组比较(P＞0.05),差别无统计学意义。 结论:酶切,PCR,测序鉴定成功地构建了CD59基因的pSUPER retro RNAi逆转录病毒载体。电镜,MTT实验及免疫组化实验显示,CD59特异位点的短肽封条具有下调survivin的表达,活化caspase-3,从而促进HeLa细胞的凋亡。我们的研究为CD59对肿瘤细胞凋亡信号转导的进一步研究奠定基础,为肿瘤的生物治疗提供一条新的思路。
[Abstract]:Objective: to construct pSUPER retro RNAi retroviral vector for CD59 gene, to lay the foundation for further research in tumor cells. CD59 RNAi uses this group of design and synthesis of the CD59 peptide seal, to detect the HeLa cell apoptosis, and provide a new way for cancer therapy.
Methods: according to the design principles for RNA interference sequence and siRNA gene of CD59 mRNA provided by GenBank and a control sequence with 19 bp fragment sequence of CD59 gene of mRNA 220-238nt as the target sequence, the oligonucleotide chain 60 at both ends was designed and synthesized with restriction sites of the plasmid pSUPER.retro.neo+GFP with the base. T_4 ligase to linear oligonucleotides were annealed and transformed into Escherichia coli JM109, plasmid was extracted. Then by enzyme digestion, PCR and sequencing of recombinant plasmid.50mg/L CD59 peptide seal in HeLa cells after 24h, apoptosis of HeLa cells by transmission electron microscopy detection; using MTT method to detect the inhibition rate of HeLa cells proliferation; using immunohistochemical method to detect survivin, Caspase-3, the expression level of Bax.
Results: after enzyme digestion, PCR and sequencing results showed that the successful construction of the pSUPER retroRNAi retroviral vector of CD59 gene. The TEM results showed that the CD59 peptide seal can make the HeLa cell nuclear.MTT fragmentation experiments show that cell proliferation of HeLa cells and short peptide CD59 gene inhibition rate more than HeLa cell + short peptide group (P < 0.01). Immunohistochemical experiments showed that HeLa + short peptide group of transfected CD59 and HeLa cells + normal peptide group, the expression level of survivin decreased, the difference was statistically significant (P < 0.05); and the expression of Caspase-3 was increased, the difference was statistically significant (P < 0.05); expression of Bax in each group (P > 0.05), the difference was not statistically significant.
Conclusion: enzyme digestion, PCR sequencing, pSUPER has been successfully constructed retro RNAi retroviral vector of CD59 gene. Electron microscope, MTT assay and immunohistochemical assay showed that the peptide seals specific sites with CD59 expression, down-regulation of Survivin and activation of Caspase-3, thereby promoting the apoptosis of HeLa cells. Our study laid the foundation for the further study of CD59 on apoptosis of tumor cell signal transduction, provide a new way for tumor biological therapy.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.12

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