人α干扰素转基因小鼠模型的建立

发布时间：2018-04-03 03:08

本文选题：干扰素　切入点：转基因小鼠　出处：《吉林大学》2011年硕士论文

【摘要】：干扰素是一种当机体受到相应诱导物刺激后由细胞分泌的具有抗病毒活性的蛋白质。干扰素通过诱导细胞内产生有酶活性的抗病毒蛋白,间接地抑制病毒复制而具有抗病毒作用;干扰素还有抗肿瘤和免疫调节作用。一般而言,当细胞受到病毒或双链RNA的刺激时就会产生IFN-α和IFN- ?。但在不同的生物体内的不同组织器官的细胞中干扰素的产生量都是不同的。大多数临床治疗中使用的干扰素都是通过基因工程产生的。前期的生物治疗研究证明,IFN-α为后来的很多新的生物治疗药物的发展铺平了道路。本研究用PvuI酶切IRES-neo-IFN质粒,回收并纯化目的基因,通过受精卵显微注射技术,将其导入到昆明小鼠受精卵的雄原核中,约注射了864枚受精卵,将823枚存活的受精卵移植到33只假孕雌鼠的输卵管壶腹部内,其中有15只怀孕,顺利出生并存活的仔鼠有31只,通过特异性引物PCR法和Southern杂交法检测出4只阳性转基因小鼠。原代转基因小鼠与正常的昆明小鼠交配,出生后代经PCR检测,筛选出F1代转基因小鼠。F1代转基因小鼠的整合率为49.3%(35/71只)。应用人IFN-α检测试剂盒对F1代PCR阳性转基因小鼠进行了体内人IFN-α蛋白表达情况的分析。收集阳性小鼠血清,用ELISA方法检测出3个转基因鼠系均有人IFN-α表达。通过本研究初步表明,我们所构建的IRES-neo-IFN表达载体不仅能够指导人IFN-α在原代小鼠中高效表达,并能够在后代中稳定的遗传和表达。本研究成功建立了表达人IFN-α基因的转基因小鼠,为干扰素抗病毒机制的研究及为其他动物进行抗病毒感染基因工程育种研究奠定了基础。
[Abstract]:Interferon is an antiviral protein secreted by cells when the body is stimulated by the corresponding inducer.Interferon has antiviral effects by inducing the production of antiviral proteins with enzyme activity, which indirectly inhibits viral replication, and also has anti-tumor and immunomodulatory effects.In general, IFN- 伪 and IFN- 伪 are produced when cells are stimulated by viruses or double-stranded RNA.But the amount of interferon produced in different tissues and organs of different organisms is different.Interferon used in most clinical treatments is produced by genetic engineering.Previous biotherapy studies have demonstrated that IFN- 伪 paved the way for the development of many new biotherapeutic drugs.In this study, the IRES-neo-IFN plasmid was digested with PvuI, and the target gene was recovered and purified. By microinjection of fertilized eggs, the plasmid was introduced into the male prokaryotic of Kunming mouse fertilized eggs, and about 864 fertilized eggs were injected.Eight hundred and twenty-three surviving fertilized eggs were transplanted into the ampulla of 33 pseudopregnant female rats. Among them, 15 were pregnant and 31 were born and survived successfully. Four positive transgenic mice were detected by specific primer PCR and Southern hybridization.The primary transgenic mice were mated with normal Kunming mice. After birth, the integration rate of F1 transgenic mice and F1 generation transgenic mice was 49.3% and 35 / 71 respectively.The expression of human IFN- 伪 protein in F1 generation PCR positive transgenic mice was analyzed by human IFN- 伪 detection kit.Positive mouse serum was collected and IFN- 伪 expression was detected by ELISA.The results showed that the constructed IRES-neo-IFN expression vector could not only direct the expression of human IFN- 伪 in primary mice, but also be stable in the inheritance and expression of human IFN- 伪 in offspring.In this study, transgenic mice expressing human IFN- 伪 gene were successfully established, which laid a foundation for the study of antiviral mechanism of interferon and genetic engineering breeding of other animals for anti-virus infection.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332

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