肠道稳定型牛腺病毒载体的构建及体外评价
发布时间：2018-08-29 18:48
【摘要】：牛腺病毒1型(BAdV-1)和3型(BAdV-3)分别为牛肠道和呼吸道常在病毒,因其本身不具致病性,生物安全性高,转导效率高,以及本身强大的递送和佐剂功能,近年来受到人们的广泛关注。在已报道的牛腺病毒中,BAdV-3研究的最为详细并已广泛用于载体疫苗研究。但是BAdV-3属呼吸道腺病毒,不耐受胃肠道的极端环境限制了其作为载体在口服疫苗研究中的应用。BAdV-1虽为嗜肠型腺病毒,具有良好的肠道稳定性,但由于其作为疫苗载体研究的相对落后,目前尚无报道的BAdV-1疫苗载体可用。考虑到牛腺病毒在开发牛口服载体疫苗领域的天然优势,本研究通过以下两种途径尝试构建具有口服疫苗载体潜力的重组BAd V-3和BAdV-1病毒。1.本研究利用BJ5183同源重组系统将BAdV-3 fiber顶端的knob区替换为BAdV-1 fiber顶端的knob区成功拯救出与wtBAdV3增殖能力相近的嵌合病毒rBAdV3-B1F,并证明该嵌合病毒可以逃逸BAdV-3阳性血清的中和。但是该重组病毒易受低pH、胃蛋白酶、胰蛋白酶和糜蛋白酶的影响,这些结果说明重组病毒的肠道稳定性并没有显著提高。2.本研究对BAdV1进行改造,通过在BAdV1的E3区引入标记基因EYFP,成功拯救出重组病毒rBAdV1-EYFP。通过PCR和Western blotting方法证明重组病毒rBAdV1-EYFP在体外能稳定传代。中和试验结果表明wtBAdV1及rBAdV1-EYFP的中和效价一致。重组病毒rBAdV1-EYFP在盐酸(pH=2)中处理之后发现,与wtBAdV1类似,重组病毒rBAdV1-EYFP可以耐受高浓度的盐酸。同时,在pH、胃蛋白酶、胰蛋白酶和糜蛋白酶混合存在的情况下,重组病毒rBAdV1-EYFP的存活率要高于野生型病毒wtBAdV1。本研究尝试构建具有肠道稳定性的牛腺病毒载体。通过对wtBAdV3的fiber蛋白的knob区进行替换,发现重组病毒的肠道稳定性并没有提高,说明这一方法不可行。我们进一步对嗜肠型牛腺病毒wtBAdV1进行改造,构建了带有标记基因EYFP的重组牛1型腺病毒rBAdV1-EYFP,并证明该重组病毒可以耐受高浓度的盐酸,这些结果为开发具有肠道稳定性的牛腺病毒疫苗载体奠定了良好的基础。
[Abstract]:Bovine adenovirus type 1 (BAdV-1) and bovine adenovirus type 3 (BAdV-3) are usually found in bovine intestinal tract and respiratory tract respectively. Because of their non-pathogenicity, high biosafety, high transduction efficiency, and their powerful delivery and adjuvant functions, bovine adenovirus type 1 (BAdV-3) has attracted wide attention in recent years. Among the reported bovine adenovirus, BAdV-3 is the most detailed and widely used in vector vaccine research. However, BAdV-3 belongs to respiratory tract adenovirus, and its application as a vector in oral vaccine research is restricted by the extreme environment of intolerant gastrointestinal tract. Although BAdV-1 is an enterotropic adenovirus, it has good intestinal stability. However, due to its relatively backward research as a vaccine vector, there is no reported BAdV-1 vaccine vector available. Considering the natural advantages of bovine adenovirus in the development of bovine oral vector vaccine, this study attempted to construct recombinant BAd V-3 and BAdV-1 virus. In this study, the BJ5183 homologous recombination system was used to replace the knob region at the tip of BAdV-3 fiber with the knob region at the top of BAdV-1 fiber, and the chimeric virus rBAdV3-B1F, was successfully saved from the BAdV-3 positive serum, which was similar to wtBAdV3's proliferative ability and proved that the chimeric virus could escape the neutralization of BAdV-3 positive serum. However, the recombinant virus is susceptible to low pH, pepsin, trypsin and chymotrypsin. These results indicate that the intestinal stability of the recombinant virus does not improve significantly. 2. In this study, BAdV1 was modified and the recombinant virus rBAdV1-EYFP. was successfully saved by introducing the marker gene EYFP, into the E3 region of BAdV1. The results of PCR and Western blotting showed that the recombinant virus rBAdV1-EYFP could be subcultured stably in vitro. Neutralization test results showed that the neutralization titers of wtBAdV1 and rBAdV1-EYFP were the same. The recombinant virus rBAdV1-EYFP was treated with hydrochloric acid (pH=2), and it was found that rBAdV1-EYFP could tolerate high concentration of HCl, similar to wtBAdV1. In the presence of pH, pepsin, trypsin and chymotrypsin, the survival rate of recombinant virus rBAdV1-EYFP was higher than that of wild-type virus wtBAdV1.. In this study, we try to construct bovine adenovirus vector with intestinal stability. By replacing the knob region of fiber protein of wtBAdV3, it was found that the intestinal stability of the recombinant virus was not improved, which indicated that this method was not feasible. We further modified the enterophilic bovine adenovirus (wtBAdV1) and constructed the recombinant bovine adenovirus type 1 (rBAdV1-EYFP,) with the marker gene EYFP and proved that the recombinant virus could tolerate high concentration of hydrochloric acid. These results laid a good foundation for the development of bovine adenovirus vaccine vector with intestinal stability.
【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】：2017
【分类号】：S852.65
本文编号：2212085
[Abstract]:Bovine adenovirus type 1 (BAdV-1) and bovine adenovirus type 3 (BAdV-3) are usually found in bovine intestinal tract and respiratory tract respectively. Because of their non-pathogenicity, high biosafety, high transduction efficiency, and their powerful delivery and adjuvant functions, bovine adenovirus type 1 (BAdV-3) has attracted wide attention in recent years. Among the reported bovine adenovirus, BAdV-3 is the most detailed and widely used in vector vaccine research. However, BAdV-3 belongs to respiratory tract adenovirus, and its application as a vector in oral vaccine research is restricted by the extreme environment of intolerant gastrointestinal tract. Although BAdV-1 is an enterotropic adenovirus, it has good intestinal stability. However, due to its relatively backward research as a vaccine vector, there is no reported BAdV-1 vaccine vector available. Considering the natural advantages of bovine adenovirus in the development of bovine oral vector vaccine, this study attempted to construct recombinant BAd V-3 and BAdV-1 virus. In this study, the BJ5183 homologous recombination system was used to replace the knob region at the tip of BAdV-3 fiber with the knob region at the top of BAdV-1 fiber, and the chimeric virus rBAdV3-B1F, was successfully saved from the BAdV-3 positive serum, which was similar to wtBAdV3's proliferative ability and proved that the chimeric virus could escape the neutralization of BAdV-3 positive serum. However, the recombinant virus is susceptible to low pH, pepsin, trypsin and chymotrypsin. These results indicate that the intestinal stability of the recombinant virus does not improve significantly. 2. In this study, BAdV1 was modified and the recombinant virus rBAdV1-EYFP. was successfully saved by introducing the marker gene EYFP, into the E3 region of BAdV1. The results of PCR and Western blotting showed that the recombinant virus rBAdV1-EYFP could be subcultured stably in vitro. Neutralization test results showed that the neutralization titers of wtBAdV1 and rBAdV1-EYFP were the same. The recombinant virus rBAdV1-EYFP was treated with hydrochloric acid (pH=2), and it was found that rBAdV1-EYFP could tolerate high concentration of HCl, similar to wtBAdV1. In the presence of pH, pepsin, trypsin and chymotrypsin, the survival rate of recombinant virus rBAdV1-EYFP was higher than that of wild-type virus wtBAdV1.. In this study, we try to construct bovine adenovirus vector with intestinal stability. By replacing the knob region of fiber protein of wtBAdV3, it was found that the intestinal stability of the recombinant virus was not improved, which indicated that this method was not feasible. We further modified the enterophilic bovine adenovirus (wtBAdV1) and constructed the recombinant bovine adenovirus type 1 (rBAdV1-EYFP,) with the marker gene EYFP and proved that the recombinant virus could tolerate high concentration of hydrochloric acid. These results laid a good foundation for the development of bovine adenovirus vaccine vector with intestinal stability.
【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】：2017
【分类号】：S852.65
【参考文献】
相关硕士学位论文 前3条
1 张潞;牛1型腺病毒感染性克隆的构建及拯救[D];西北农林科技大学;2015年
2 马静;小衣壳蛋白pⅨ对3型牛腺病毒包装作用的研究[D];西北农林科技大学;2014年
3 于作;牛腺病毒3型中国分离株的鉴定及全基因组序列分析[D];中国农业科学院;2011年
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