门静脉注射小鼠Hepcidin基因特异性RNAi腺病毒的方法研究

发布时间：2018-04-06 23:32

本文选题：Hepcidin　切入点：腺病毒科　出处：《浙江大学》2013年硕士论文

【摘要】：研究目的：构建含Hepcidin基因重组腺病毒干扰真核表达载体Pad-hepc-shRNA-GFP,并验证其门静脉注射途径的干预效果。实验方法： 1) Pad-hepc-shRNA-EGFP的构建：根据Hepcidin基因在genbank的序列,设计相应的siRNA,通过设计构架oligo,合成shRNA序列。设计并合成1对互补的含shRNA序列的寡聚单链DNA,按一定的体系退火形成DNA双链,然后用载体构建试剂盒BLOCK-iT U6RNAi Entry Vector Kit (invitrogen, Catalog nos. K4944-00)进行重组克隆,将退火而成的双链shRNA oligo插入shRNA表达载体pENTR/U6vector中,按照一定的连接体系,构建shRNA表达质粒,并转化至感受态细胞DH5a孵育,小量质粒抽提并且测序。设计引物、运用特定的酶切体系,对于构建的pENTR/U6载体进行改造,连入CMV启动子和荧光蛋白GFP,然后使用Invitrogen公司的LR重组系统将改造好的载体和pAd/BLOCK-IT-DEST重组,转化至感受态细胞DH5α孵育,筛选并测序,验证Pad-hpec-shRNA-EGFP(?)建成功。用构建的腺病毒载体Pad-hepc-shRNA-EGFP经Pac I消化后转染293A细胞,包装病毒,收集初级病毒液,二次扩增,收集病毒原液并测定滴度,备用。 2)经小鼠门静脉注射干扰病毒：对正常balb/c小鼠麻醉后,行门静脉注射载体病毒量2.7109ifu/ml/500ul,正常喂养10d后,取肝脏标本,通过半定量PT-PCR法检测小鼠肝脏Hepcidin基因的表达情况。对照组取正常小鼠肝脏标本检测Hepcidin基因的表达情况。研究结果：经过基因测序证明,腺病毒重组载体Pad-hepc-shRNA-EGFP中包含的基因片段与目的基因一致,成功构建了含Hepcidin基因重组腺病毒干扰真核表达载体,经过包装、扩增,并获得了滴度为2.7109ifu/ml腺病毒液。病毒门静脉注射后,实验组小鼠肝脏Hepcidin mRNA表达为1.30±0.53,明显低于对照组(6.39±1.00),充分显示出了门静脉注射此载体能够对Hepcidin基因的有效的抑制。结论：门静脉注射Hepcidin基因特异性重组腺病毒干扰载体Pad-hepc-shRNA-EGFP能够有效抑制小鼠Hepcidin基因的表达。
[Abstract]:Objectives of the study:The recombinant adenovirus interference eukaryotic expression vector Pad-hepc-shRNA-GFPFP containing Hepcidin gene was constructed and its effect on portal vein injection was verified.Experimental methods:1) the construction of Pad-hepc-shRNA-EGFP: according to the sequence of Hepcidin gene in genbank, the corresponding siRNAs were designed, and the shRNA sequence was synthesized by the framework of oligo.A pair of complementary oligonucleotides containing shRNA sequences were designed and synthesized, and DNA double strands were synthesized by annealing in a certain system. Then the kit BLOCK-iT U6RNAi Entry Vector Kit was constructed by using vector to construct BLOCK-iT U6RNAi Entry Vector Kit invitrogen, Catalog nos.K4944-00) was cloned into the shRNA expression vector pENTR/U6vector by inserting the annealed double-stranded shRNA oligo. According to a certain ligation system, the shRNA expression plasmid was constructed. The shRNA expression plasmid was transformed into the receptive cell DH5a incubated, and a small number of plasmids were extracted and sequenced.Primer was designed, and the constructed pENTR/U6 vector was modified with specific enzyme digestion system. The recombinant vector was linked to CMV promoter and fluorescent protein GFP. then the transformed vector and pAd/BLOCK-IT-DEST were recombined by LR recombination system of Invitrogen Company, and then transformed to DH5 伪 cells.Pad-hpec-shRNA-EGFPN was screened and sequenced to verify Pad-hpec-shRNA-EGFP.The building was successful.The recombinant adenovirus vector Pad-hepc-shRNA-EGFP was digested by Pac I and transfected into 293A cells. The virus was packaged, the primary virus solution was collected, the virus was amplified twice, the titer of the virus was collected and the titer was measured.2) injecting interfering virus through portal vein of mice: after anaesthesia to normal balb/c mice, the volume of vector virus was 2.7109 ifuml / r / 500ul.After normal feeding for 10 days, the liver samples were taken and the expression of Hepcidin gene in the liver of mice was detected by semi-quantitative PT-PCR method.The expression of Hepcidin gene in normal mouse liver was detected in the control group.Results of the study:It was proved by gene sequencing that the recombinant adenovirus vector Pad-hepc-shRNA-EGFP contained the same gene fragment as the target gene. The recombinant adenovirus containing Hepcidin gene interference eukaryotic expression vector was successfully constructed. After packaging, amplification, and titer of 2.7109ifu/ml adenovirus solution was obtained.After portal vein injection, the expression of Hepcidin mRNA in liver of experimental group was 1.30 卤0.53, which was significantly lower than that of control group (6.39 卤1.00), which showed that portal vein injection of the vector could effectively inhibit the expression of Hepcidin gene.Conclusion:Portal vein injection of Hepcidin gene specific recombinant adenovirus interference vector Pad-hepc-shRNA-EGFP can effectively inhibit the expression of mouse Hepcidin gene.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7

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