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缺失E1区猪3型腺病毒包装细胞系的构建

发布时间:2018-01-21 12:26

  本文关键词: 猪3型腺病毒 慢病毒 稳定细胞系 病毒增殖与包装 出处:《西北农林科技大学》2015年硕士论文 论文类型:学位论文


【摘要】:猪3型腺病毒(PAdV-3)于1964年从健康猪肠道首次分离。在猪用口服疫苗研究方面,PAdV-3在肠道嗜性、免疫原性和逃逸自身载体免疫方面,显著优于目前普遍使用的人5型腺病毒(HAdV-5)。此外,在人用疫苗研究方面,由于PAdV-3与HAdV-5无血清交叉反应,避免了人体普遍存在HAdV-5载体免疫的干扰和造成潜在的组织损伤,因此PAdV-3比HAdV-5也更具安全性。充分发挥PAdV-3以上优势的前提是具备缺失E1区猪3型腺病毒(PAdV-3ΔE1)的感染性克隆和高效包装细胞系。本实验室目前已拥有PAdV-3ΔE1感染性克隆,但是由于已报道包装PAdV-3ΔE1的细胞系VR1BL(稳定表达HAdV-5 E1和PAdV-3 E1Blarge的猪视网膜上皮细胞系)受到多达13项专利保护,因此,基于PAdV-3ΔE1的口服疫苗及基因递送系统研究受到了极大的限制。考虑到野生型PAdV-3除了可在VR1BL中增殖外,还可在猪睾丸(swine testicular,ST)细胞中高效增殖,本研究基于第二代重组慢病毒包装系统,构建可稳定表达PAdV-3 E1Blarge和I-SceI的细胞系——ST-E1-E1Blarge-ISceI。与专利保护的VR1BL细胞相比,ST-E1-E1Blarge-ISce I细胞可实现PAdV-3ΔE1的成功包装和高效增殖,病毒包装能力略低于VR1BL细胞,但96 h内病毒增殖能力比VR1BL细胞高100倍。本研究的主要内容和结果如下:1.第二代重组慢病毒的包装构建第二代慢病毒包装用克隆载体。在本实验室已构建的克隆载体的基础上,合成连接肽3HA-MCS1-2A-MCS2,将合成片段连接到克隆载体上构建成pTrip-CMV-3HA-MCS1-2A-MCS2-IRES-Hygro。在MSC1处插入3NLS-ISceI基因,在MSC2处插入His-E1Blarge基因序列。PCR、酶切鉴定及测序正确的载体,按照操作步骤将包装载体psPAX2、病毒衣壳载体pMD2.G、克隆载体共转染293T细胞,包装慢病毒后测定病毒滴度。进而从慢病毒抗性筛选和蛋白表达两个方面验证重组慢病毒包装成功。2.ST-E1-E1Blarge-ISceI细胞系的筛选10 MOI重组慢病毒转导ST-E1细胞,按照标准操作步骤进行潮霉素筛选细胞,连续筛选3轮后,挑取细胞单克隆传代培养,分别收集第5代、第10代和第30代细胞样品,用Western blot方法检测细胞中是否稳定表达PAdV-3 E1Blarge和I-SceI,分别用抗HA鼠单抗和抗His鼠单抗作为一抗。实验结果表明,ST-E1-E1Blarge-ISceI细胞筛选成功并能稳定表达PAdV-3 E1Blarge和I-SceI。3.检测PAdV-3ΔE1在VR1BL和ST-E1-E1Blarge-ISceI上拯救和增殖能力将本实验室已有的PAd V-3ΔE1基因组载体,经Pac I酶切线性化,使用Lipofectin reagent转染VR1BL和ST-E1-E1Blarge-ISceI细胞,包装E1区表达绿色荧光的PAdV-3病毒(PAdV219),观察转染效率和测定包装病毒的滴度。实验结果表明,本实验室筛选的细胞可以包装PAd V-3ΔE1,但病毒基因组转染效率和病毒包装效率略低于VR1BL细胞。在病毒增殖能力方面,72 h内本文构建细胞与VR1BL细胞具有相似增殖能力。但在72-96 h之间,由于本文构建细胞存活率仍接近50%,而VR1BL细胞全部脱落死亡,所以本文构建细胞病毒增殖滴度比VR1BL细胞高出100倍。综合以上实验结果表明,本实验室筛选细胞系具有高效PAdV-3ΔE1包装和增殖能力,并有望极大促进基于PAdV-3ΔE1口服载体疫苗和基因递送系统的研究。
[Abstract]:Porcine adenovirus type 3 (PAdV-3) in 1964 for the first time isolated from healthy pigs. The pigs in the intestinal tract of the oral vaccine, eosinophilic intestinal PAdV-3, immunogenicity and immune escape its carrier, significantly better than that of the human adenovirus type 5 (HAdV-5). In addition, people used in vaccine research no, because the serum cross reaction of PAdV-3 and HAdV-5, to avoid the human universal vector of HAdV-5 interference and immune injury potential, so PAdV-3 is more than HAdV-5. The premise of safety and give full play to PAdV-3 advantage is above E1 deletion region of porcine adenovirus type 3 (PAdV-3 E1) and the infectious clone efficient packaging cell line. The laboratory now has PAdV-3 E1 infectious clone, but because VR1BL cells have been reported PAdV-3 packaging E1 Delta (stable pig retinal pigment epithelial cell line expressing HAdV-5 E1 and PAdV-3 E1Blarge) by up to 1 3 items of patent protection, therefore, oral vaccine and gene delivery system of PAdV-3 E1 has been greatly restricted. Based on the consideration of wild type PAdV-3 in addition to the proliferation in VR1BL, but also in the pig testis (swine testicular, ST), the proliferation of cells, second generation recombinant lentiviral packaging system based on this research construction of ST-E1-E1Blarge-ISceI. cells, and the patent protection system with stable expression of PAdV-3 E1Blarge and I-SceI VR1BL cells compared to I cells, ST-E1-E1Blarge-ISce can achieve the success of the E1 PAdV-3 delta packaging and high proliferation, virus packaging capacity is slightly lower than that of VR1BL cells, but the 96 h virus proliferation of VR1BL cells was 100 times higher than the main content and results of this. The research is as follows: 1. the second generation of recombinant lentivirus packaging construction of the second generation lentivirus packaging cloning vector. Based on cloning vectors have been constructed in our lab on a connection The synthetic peptide 3HA-MCS1-2A-MCS2, cloned into cloning vector to construct pTrip-CMV-3HA-MCS1-2A-MCS2-IRES-Hygro. 3NLS-ISceI gene in MSC1, insert the His-E1Blarge.PCR gene sequence in MSC2, enzyme digestion identification and sequencing vector, according to the operation steps of loading package psPAX2, the viral capsid pMD2.G vector cloning vector were transfected into 293T cells. The viral titer was determined by packaging slow virus. And then screened from slow virus resistance and protein expression in two aspects to verify the recombinant lentiviral packaging cell line.2.ST-E1-E1Blarge-ISceI successfully screened 10 MOI recombinant lentiviral transduction of ST-E1 cells, in accordance with the standard operating procedure of hygromycin screening cells, continuous screening after the 3 round, picked the cell monoclonal passaging, were collected from fifth generation. The tenth and thirtieth generation cell samples, using Western blot method to detect whether cells with stable expression of PAdV-3 E1Bla Rge and I-SceI, respectively, with anti HA monoclonal antibody and anti mouse His monoclonal antibody as the first antibody. The experimental results show that ST-E1-E1Blarge-ISceI cells successfully screened and stably expressed PAdV-3 E1Blarge and I-SceI.3. PAdV-3 detection in VR1BL and ST-E1-E1Blarge-ISceI E1 to save on the proliferation and PAd V-3 E1 genome vector of this laboratory has, by Pac I were linearized with Lipofectin reagent transfected VR1BL and ST-E1-E1Blarge-ISceI cells, green fluorescent virus expressing PAdV-3 E1 region (PAdV219), packaging and packaging to observe the transfection efficiency determination of virus titer. The experimental results show that the laboratory screening cells can PAd V-3 a E1 package, but the transfection efficiency of virus genome and virus packaging efficiency slightly lower than that of VR1BL cells. The virus proliferation, we construct 72 h cells and VR1BL cells have similar proliferation ability. But in the 72-96 h, because this Construction of cell survival rate is close to 50%, while the VR1BL cell death all off, so the construction of cell proliferation of virus titer is 100 times higher than that of VR1BL cells. The above results show that the laboratory screening of cell lines with high PAdV-3 E1 packaging and proliferation, and is expected to greatly promote the research of PAdV-3 E1 oral vaccine and gene carrier based on the delivery system.

【学位授予单位】:西北农林科技大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:S852.65

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