CN106362145A - 一种h5亚型禽流感dna疫苗的构建方法 - Google Patents

一种h5亚型禽流感dna疫苗的构建方法 Download PDF

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CN106362145A
CN106362145A CN201610856289.6A CN201610856289A CN106362145A CN 106362145 A CN106362145 A CN 106362145A CN 201610856289 A CN201610856289 A CN 201610856289A CN 106362145 A CN106362145 A CN 106362145A
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porcine
protein gene
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avian influenza
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蒋文明
李金平
侯广宇
朱琳
陈继明
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CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER
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Abstract

本发明属于生物技术领域，确立了一种H5亚型禽流感病毒DNA疫苗的构建方法，利用猪捷申病毒1型（PTV‑1）的2A蛋白或口蹄疫病毒（FMDV）的2A蛋白具有自裂解功能，将Re‑6、Re‑7、Re‑8疫苗病毒HA1基因经猪捷申病毒1型（PTV‑1）的2A蛋白基因或口蹄疫病毒（FMDV）的2A蛋白基因串联，插入到真核表达质粒中。该重组质粒可以分别表达Re‑6、Re‑7、Re‑8疫苗病毒的HA1蛋白。制备成DNA疫苗后，免疫动物可以同时抵抗三种流行分支H5亚型禽流感病毒的攻击，在生产上有较大的开发和应用价值。

Description

一种H5亚型禽流感DNA疫苗的构建方法
技术领域
本发明属于生物技术领域；具体说来，本发明确立了一种构建H5亚型禽流感DNA疫苗的方法，用于H5亚型禽流感的免疫与防控，在生产上有较大的开发和应用价值。
背景技术
禽流感是由禽流感病毒引起的一种禽类传染病。禽流感病毒根据血凝素（HA）和神经氨酸酶（NA）的不同，可分为16个HA亚型和9个NA亚型。高致病性禽流感是由某些H5或H7亚型禽流感病毒引起的严重威胁禽类的烈性传染病。
2003年以来，H5亚型高致病性禽流感一直在包括中国在内的多个国家和地区流行，给养禽业造成了严重的经济损失。目前，疫苗仍然是防控该病的重要手段。2005年，我国开始实施针对禽流感的大规模强制性免疫政策，并取得了显著成效。目前，我们国家流行的H5亚型禽流感主要为第2.3.2.1分支、第7.2分支和第2.3.4.4分支的病毒，与之对应的这3个分支的疫苗分别为Re-6、Re-7和Re-8疫苗。目前市场上主要使用的是Re-6+Re-7+Re-8三价苗或Re-6+Re-8二价苗，这给疫苗生产和使用都造成了不便。猪捷申病毒1型（PTV-1）的2A蛋白和口蹄疫病毒（FMDV）的2A蛋白具有自裂解功能，可以将串联的蛋白裂解成完整的蛋白个体。本专利利用2A蛋白的这个特性，提供了一种构建H5亚型禽流感病毒DNA疫苗的方法。
发明内容
本发明研究建立了一种构建H5亚型禽流感病毒疫苗的方法。首先利用PCR或基因合成的方法，将Re-6疫苗病毒的HA1蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白基因或口蹄疫病毒（FMDV）的2A蛋白基因、Re-7疫苗病毒的HA1蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白基因或口蹄疫病毒（FMDV）的2A蛋白基因、Re-8疫苗病毒的HA1蛋白基因（含终止密码子）五者串联，然后插入到真核表达载体pCAGGS中，构建成重组真核表达质粒，制备成DNA疫苗。该DNA疫苗可以分别表达出Re-6、Re-7、Re-8疫苗病毒的HA1蛋白。
具体实施方式
下面通过实施例，说明本发明的技术方案，但本发明的保护范围不限于这个实施例。
本实施例利用基因合成技术，人工合成将Re-6疫苗病毒的HA1蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白基因、Re-7疫苗病毒的HA1蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白基因、Re-8疫苗病毒的HA1蛋白基因（含终止密码子）五者串联体，插入到真核表达载体pCAGGS中，构建成重组真核表达质粒。将重组质粒转染293T细胞，分别使用针对Re-6、Re-7、Re-8疫苗的阳性抗体进行蛋白免疫印迹试验，可以检测到Re-6、Re-7、Re-8疫苗病毒的HA1蛋白的表达。
将该重组质粒制备成DNA疫苗，免疫3周龄SPF鸡，100μg/只，免疫4周后，分别用第2.3.2.1分支、第7.2分支和第2.3.4.4分支的H5强毒攻毒，能够完全保护SPF鸡抵抗病毒的攻击，免疫攻毒试验鸡不发病、不排毒、不死亡。

Claims (4)

1.利用PCR或基因合成的方法，将Re-6疫苗病毒的HA1蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白基因或口蹄疫病毒（FMDV）的2A蛋白基因、Re-7疫苗病毒的HA1蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白基因或口蹄疫病毒（FMDV）的2A蛋白基因、Re-8疫苗病毒的HA1蛋白基因（含终止密码子）五者串联，插入到真核表达载体中，构建成重组真核表达质粒，制备DNA疫苗。
2.如权利要求1所述的方法，构建重组真核表达质粒，用作H5亚型禽流感的免疫。
3.如权利要求1所述的方法，利用猪捷申病毒1型的2A蛋白，构建表达多个保护性抗原蛋白的重组真核表达质粒，用作相关疫病的免疫。
4.如权利要求2和3所述的重组真核表达质粒，用于疫苗的生产。
CN201610856289.6A 2016-09-28 2016-09-28 一种h5亚型禽流感dna疫苗的构建方法 Pending CN106362145A (zh)

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CN101380468A (zh) * 2008-07-17 2009-03-11 浙江省农业科学院 猪繁殖与呼吸综合征二价重组腺病毒疫苗及其制备方法
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