导入β-半乳糖苷酶基因的重组腺病毒的纯化

发布时间：2018-01-12 02:16

  本文关键词：导入β-半乳糖苷酶基因的重组腺病毒的纯化　出处：《河北医科大学》2012年硕士论文　论文类型：学位论文

  更多相关文章： 腺病毒 LacZ基因 转染 人肾胚293细胞 纯化

【摘要】：目的：伴随工业、农业和交通事业的发展，高能量创伤日益增多，周围神经损伤已成为临床上比较常见的致残性损伤，而针对周围神经损伤的治疗一直是临床上比较棘手的问题，尽管显微外科修复技术已经十分精湛，但长期以来难获得满意效果。随着分子生物及基因工程的发展，当今针对基因治疗研究已成为周围神经损伤研究领域的热点课题。通过研究发现在中枢和周围神经系统中把携带目的基因的腺病毒作为载体(Adenovirus,AdV)导入后，目的基因就会适时地表达产物，从而达到治疗或者研究的目的。 其中在介导基因转染方面腺病毒载体具有诸多优点，因此在神经示踪研究、神经再生和神经运动系统疾病的诊治中成为应用最广泛的载体。在神经系统内导入以腺病毒作为载体的目的基因，通过目的基因在神经系统内的表达促进周围神经的再生，同时也对神经示踪的研究的具有重要意义。 当下，神经修复研究领域的热点是将各种神经营养因子基因融合到复制缺陷型重组腺病毒（Adenovirus,AdV）中，再将携带目的基因的腺病毒转入到脊髓或周围神经中来对受损的神经元进行保护或者在周围神经系统中诱导周围神经的再生过程。腺病毒载体具有较高的感染效率、广泛的宿主范围、可以将分裂期或静息期细胞感染、巨大的基因装载量、容易提取高浓度病毒成分、毒性低水平表达等优点。利用分子生物技术克隆乳糖操纵子基因片段LacZ基因构建腺病毒载体，制备病毒颗粒从而为进一步研究腺病毒载体介导的LacZ基因在周围神经损伤中的应用。 LacZ基因广泛用于基因表达调控研究中的一种基因。LacZ基因编码的beta一半乳糖普酶（简称beta-gal)是由4个亚基组成的四聚休，，可催化乳糖的水解．Beta-gal比较稳定，用X-Gal为底物进行染色时，呈蓝色，便于检测和观察．LacZ基因的诸多优点使它成为基因工程实验中的一个常用标记基因，因此它被用于基因的时空表达、探测已知调控序列有效区段的位置和作用、寻找未知序列等领域。 因此，利用腺病毒安全、稳定、高效以及LacZ基因特异性表达等特点当下利用含LacZ基因的腺病毒对周围神经损伤修复进行研究已成为基因治疗的前沿。但腺病毒可以被中和抗体迅速地清除，这是由于腺病毒载体的免疫原性比较强，在日常工作生活中腺病毒侵入人体后会诱发机体产生抗腺病毒中和抗体。如果接受腺病毒载体介导的基因治疗后因为中和抗体会消灭腺病毒载体，从而导致腺病毒载体在体内的数量减少而使基因治疗失败。为了达到持续时间长、表达效果确切等效果，本实验将已有腺病毒通过转染至293细胞，通过在293细胞中的繁殖，从而提取出具有侵染力的浓缩的高纯度病毒。为下一步和今后研究将LacZ基因导入AdV后在中枢和周围神经系统内的表达来促进周围神经的再生奠定基础。 方法： 1HEK293细胞的培养把冻存的细胞由-196℃的液氮中快速放入37℃水浴中融化，放入后缓慢摇晃冻存管，使细胞外冰晶融化。3000r/min离心3min融化好的细胞悬液。将上层的上清液吸去，并放入10ml培养液到离心管中，轻轻吹打成悬液，放入细胞培养瓶中。将盛有细胞和培养液培养瓶在培养箱中培养。培养箱的环境接近人体环境，为37℃和5％CO2。当细胞生长均匀时，便可计数一定体积细胞悬液中的细胞数。然后根据公式换算出细胞在每毫升细胞悬液中含有的量。细胞生长到占瓶底面积的90％还需要2-3次传代培养。显微镜下观察细胞，挑选形状饱满、生长能力强的细胞接种病毒。 2病毒转染把293细胞放入75cm2培养瓶中后加入10mlDMEM5％进行培养。待细胞生长至60%-70%时将培养液倒去，小心加入病毒混合液。十字形轻轻晃动培养瓶使培养液盖住细胞层。放入培养箱中培养90分钟后加入9mlDMEM5％，再经过72小时孵育，便可以通过MOI测定来判断病毒颗粒。 3腺病毒扩增将细胞在-20℃到37℃分别放置半小时，反复三次。用离心管离心后收集上清液冰冻存于-20℃或-80℃。取三瓶培养理想的293细胞，倒掉里面的细胞培养液，将5ml混合液放到每个培养瓶中，轻轻晃动混匀。放到培养箱中孵育，时间大约为90分钟。向培养瓶中加入DMEM，继续培养，时间大约48-72小时。同样的方法从第一次被转染的细胞中取上清后再次转染细胞。便可得到再次扩增的病毒。 4病毒的纯化离心细胞，待细胞碎片沉淀后取上清液。5mlPEG8000放入每10ml上清液中，将上清放到冰上1小时使其沉淀。以12000rpm的速度离心上述混合物，时间大约20min。将上清液倒去后，在CsCl溶液中加入沉淀物。经过5分钟离心，收集病毒悬浮液。 550%组织培养感染剂量法测定病毒滴度取293细胞一瓶，用2%FCS/DMEM将细胞浓度调整为为7.5×105/ml；将100μl细胞悬液分别放到2块96孔板中。10孔为一个稀释度，其中2孔作为阴性对照；经过10天培养后镜下观察，数出每一排出现细胞病变现象（CPE）的孔的数目。并利用Karber方程计算滴度T=101+d(s-0.5)，测出AdLacZ病毒液的TCID50：1010TCID50/ml。 结果： 1正常具有活力的HEK293细胞呈多角形，贴壁较疏松，细胞之间相互连成网状，并有聚集成团的倾向。 2含lacz基因的腺病毒转染后的293细胞在培养24小时后即可在倒置显微镜下观察到细胞变性即CPE现象而且通过Ｘ－ｇａｌ染色后可观察到细胞出现蓝染，即检测到报告基因lacz的表达，细胞表达出携带目的基因的腺病毒颗粒，对照组未发现蓝染表达。表明转染成功。 3在96孔板上培养293细胞，用获得病毒感染293细胞后计数出现CPE孔个数，测定病毒滴度。最后获得病毒滴度为1010TCID50/ml（1×109pfu/ml）。 结论：成功的纯化了AdLacZ腺病毒，并通过提高了病毒液滴度，为周围神经再生机制的研究及神经损伤的基因治疗奠定基础。利用50%组织培养感染剂量法测定病毒滴度，最终得到滴度为1010TCID50/ml（1×109pfu/ml）的病毒液，为进一步实验开辟了道路。
[Abstract]:Objective: with the development of industry, agriculture and transportation, high energy trauma is increasing, peripheral nerve injury has become a common clinical disabling injury, and for the treatment of peripheral nerve injury has been a clinical difficult problem, although microsurgical repair technique is superb, but for a long time is difficult to obtain satisfactory results. With the rapid development of molecular biological and genetic engineering, the study of gene therapy has become a hot topic in the research field of peripheral nerve injury. The study found that the target gene with adenovirus as a vector in the central and peripheral nervous system (Adenovirus, AdV) after the introduction, the purpose of gene expression product in a timelymanner, so to achieve the purpose of treatment or research.
In the aspect of gene transfection mediated by adenovirus vector has many advantages, so in the study of neural tracer, becomes the carrier of the most widely used in diagnosis and treatment of nerve regeneration and nerve system diseases. In the nervous system to import adenovirus as a gene vector, the gene expression in the nervous system to promote the regeneration of peripheral nerve at the same time, research on neural tracing is of great significance.
At present, the hot research in the field of nerve repair is the fusion of various neurotrophic factor gene by recombinant adenovirus (Adenovirus, AdV), then the adenovirus carrying purpose gene transferred to the spinal cord or peripheral nerve to protect or damaged neurons induced by peripheral nerve regeneration process in the peripheral nervous system. The infection efficiency of adenovirus vector with high, wide host range, can be split or resting cell infection, a huge amount of genetic load, easy to extract high concentration of viral components, has the advantages of low toxicity level expression. Construction of recombinant adenovirus vector using molecular biotechnology cloning lactose operon fragment of LacZ gene, preparation the virus particles so as to further study the application of LacZ gene mediated by adenovirus in peripheral nerve injury.
LacZ gene is widely used in gene expression of a gene encoding.LacZ gene regulation of beta in half lactose Cape enzyme (beta-gal) is composed of 4 subunits of four poly Hugh,.Beta-gal can catalyze the hydrolysis of lactose is relatively stable, the substrate X-Gal staining, blue, the advantages of convenient detection and observation the.LacZ gene to make it become a commonly used marker gene engineering experiments, so it is used for gene expression time, position and function of detecting effective segments of known regulatory sequences, for unknown sequences.
Therefore, the use of adenovirus is safe, stable, efficient and LacZ gene specific expression characteristics of the current use of adenovirus with LacZ gene on the repair of peripheral nerve injury has become the forefront of gene therapy. But adenovirus neutralizing antibodies can be removed quickly, this is the immunogenicity in adenovirus vector is relatively strong human adenovirus, invasion in daily life can induce neutralizing anti adenovirus antibody. If gene therapy mediated by adenovirus vector because the neutralizing antibody after elimination of adenovirus vector, resulting in adenovirus vector reduced gene therapy in vivo. The number of failed to achieve lasting for a long time, the expression of the exact effect and other effects, this experiment will have been transfected to 293 cells by adenovirus, through in 293 cell reproduction, in order to extract high purity concentrated with the infection of the disease Poison. It lays the foundation for the next and future research to promote the regeneration of the peripheral nerve after introducing the LacZ gene into AdV in the central and peripheral nervous system.
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