腸道穩(wěn)定型牛腺病毒載體的構建及體外評價
發(fā)布時間：2018-08-29 18:48
【摘要】：牛腺病毒1型(BAdV-1)和3型(BAdV-3)分別為牛腸道和呼吸道常在病毒,因其本身不具致病性,生物安全性高,轉導效率高,以及本身強大的遞送和佐劑功能,近年來受到人們的廣泛關注。在已報道的牛腺病毒中,BAdV-3研究的最為詳細并已廣泛用于載體疫苗研究。但是BAdV-3屬呼吸道腺病毒,不耐受胃腸道的極端環(huán)境限制了其作為載體在口服疫苗研究中的應用。BAdV-1雖為嗜腸型腺病毒,具有良好的腸道穩(wěn)定性,但由于其作為疫苗載體研究的相對落后,目前尚無報道的BAdV-1疫苗載體可用�？紤]到牛腺病毒在開發(fā)�？诜d體疫苗領域的天然優(yōu)勢,本研究通過以下兩種途徑嘗試構建具有口服疫苗載體潛力的重組BAd V-3和BAdV-1病毒。1.本研究利用BJ5183同源重組系統(tǒng)將BAdV-3 fiber頂端的knob區(qū)替換為BAdV-1 fiber頂端的knob區(qū)成功拯救出與wtBAdV3增殖能力相近的嵌合病毒rBAdV3-B1F,并證明該嵌合病毒可以逃逸BAdV-3陽性血清的中和。但是該重組病毒易受低pH、胃蛋白酶、胰蛋白酶和糜蛋白酶的影響,這些結果說明重組病毒的腸道穩(wěn)定性并沒有顯著提高。2.本研究對BAdV1進行改造,通過在BAdV1的E3區(qū)引入標記基因EYFP,成功拯救出重組病毒rBAdV1-EYFP。通過PCR和Western blotting方法證明重組病毒rBAdV1-EYFP在體外能穩(wěn)定傳代。中和試驗結果表明wtBAdV1及rBAdV1-EYFP的中和效價一致。重組病毒rBAdV1-EYFP在鹽酸(pH=2)中處理之后發(fā)現(xiàn),與wtBAdV1類似,重組病毒rBAdV1-EYFP可以耐受高濃度的鹽酸。同時,在pH、胃蛋白酶、胰蛋白酶和糜蛋白酶混合存在的情況下,重組病毒rBAdV1-EYFP的存活率要高于野生型病毒wtBAdV1。本研究嘗試構建具有腸道穩(wěn)定性的牛腺病毒載體。通過對wtBAdV3的fiber蛋白的knob區(qū)進行替換,發(fā)現(xiàn)重組病毒的腸道穩(wěn)定性并沒有提高,說明這一方法不可行。我們進一步對嗜腸型牛腺病毒wtBAdV1進行改造,構建了帶有標記基因EYFP的重組牛1型腺病毒rBAdV1-EYFP,并證明該重組病毒可以耐受高濃度的鹽酸,這些結果為開發(fā)具有腸道穩(wěn)定性的牛腺病毒疫苗載體奠定了良好的基礎。
[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.
【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65
本文編號：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.
【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65
【參考文獻】
相關碩士學位論文 前3條
1 張潞;牛1型腺病毒感染性克隆的構建及拯救[D];西北農林科技大學;2015年
2 馬靜;小衣殼蛋白pⅨ對3型牛腺病毒包裝作用的研究[D];西北農林科技大學;2014年
3 于作;牛腺病毒3型中國分離株的鑒定及全基因組序列分析[D];中國農業(yè)科學院;2011年
，本文編號：2212085
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