CN106399265B - 一种提高禽流感疫苗毒株增殖滴度的方法 - Google Patents

一种提高禽流感疫苗毒株增殖滴度的方法 Download PDF

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CN106399265B
CN106399265B CN201610829472.7A CN201610829472A CN106399265B CN 106399265 B CN106399265 B CN 106399265B CN 201610829472 A CN201610829472 A CN 201610829472A CN 106399265 B CN106399265 B CN 106399265B
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avian influenza
influenza vaccine
recombinant virus
viral titer
vaccine strain
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CN106399265A (zh
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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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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

本发明属于生物技术领域，用于提高Re‑7禽流感疫苗毒株的增殖滴度。本发明应用定点突变技术，将Re‑7疫苗毒株的HA蛋白的第171为氨基酸进行突变，再利用反向遗传操作技术重新获得重组病毒，能够显著提高Re‑7疫苗毒株的增殖滴度，从而大大降低生产成本。

Description

一种提高禽流感疫苗毒株增殖滴度的方法
技术领域
本发明属于生物技术领域；具体说来，本发明确立了一种提高H5亚型禽流感疫苗毒株增殖滴度的方法，用于疫苗的生产，降低生产成本，在生产上有较大的使用价值。
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。随着病毒的不断变异，疫苗也从Re-1更新到目前的Re-6、Re-7、Re-8。生产中发现，Re-7在鸡胚上的增殖滴度要明显低于Re-6和Re-8的滴度。这就使得Re-7疫苗生产的成本要高于Re-6和Re-8疫苗的生产成本。本专利提供了一种提高Re-7疫苗毒株增殖滴度的方法。
发明内容
本发明针对上述Re-7疫苗毒株增殖滴度的不足，研究建立了一种提高Re-7疫苗毒株在鸡胚上增殖滴度的方法。首先利用定点突变技术，将Re-7疫苗毒株的HA基因进行突变，使其编码的第171位氨基酸突变为异亮氨酸（I）或缬氨酸（V），再利用反向遗传操作技术重新获得重组病毒。将重组病毒接种鸡胚，能够显著提高Re-7疫苗毒株的增殖滴度。
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。
本实施例利用定点突变技术，对Re-7禽流感疫苗毒株HA基因进行点突变，使其编码的第171位氨基酸由原来的苏氨酸（T）突变为异亮氨酸（I）或缬氨酸（V），再利用反向遗传操作技术重新获得重组病毒。将突变之前的Re-7疫苗株和突变后的重组病毒接种10日龄SPF鸡胚，37°C孵化72小时，其HA滴度分别达到9和11，突变后的重组病毒比突变之前的疫苗毒株提高了2个HA滴度。

Claims (3)

1.一种获得禽流感重组病毒的方法，其特征在于，所述方法采用利用定点突变技术，对Re-7禽流感疫苗毒株HA基因进行点突变，使其编码的第171位氨基酸由原来的苏氨酸(T)突变为异亮氨酸(I)或缬氨酸(V)，再利用反向遗传操作技术重新获得重组病毒。
2.如权利要求1所述的方法得到的重组病毒，用作禽流感疫苗生产的种毒。
3.如权利要求1所述的方法得到的重组病毒，用于禽流感疫苗的生产。
CN201610829472.7A 2016-09-19 2016-09-19 一种提高禽流感疫苗毒株增殖滴度的方法 Expired - Fee Related CN106399265B (zh)

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CN108913701B (zh) * 2018-08-13 2020-08-28 中国动物卫生与流行病学中心 一种提高h7n9亚型禽流感疫苗毒株增殖滴度的方法

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