Adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes and preparation method of adenovirus vector bivalent vaccine

A bird flu and adenovirus technology, applied in the direction of virus/bacteriophage, chemical instruments and methods, botany equipment and methods, etc., can solve the problem of single function and achieve the effect of high environmental pressure, long production cycle and poor safety

Active Publication Date: 2020-12-22
河北省动物疫病预防控制中心 +1
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Problems solved by technology

However, the disadvantage of this vaccine is that it can only be specifically immunized against one avian influenza H7 subtype virus, has a single function, and cannot produce immune protection against subtypes of avian influenza infections such as H5 and H9; according to authoritative epidemiological

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  • Adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes and preparation method of adenovirus vector bivalent vaccine
  • Adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes and preparation method of adenovirus vector bivalent vaccine
  • Adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes and preparation method of adenovirus vector bivalent vaccine

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preparation example Construction

[0040] The invention provides a method for preparing an adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes, comprising the following steps:

[0041] S1, preparation of plasmid pUC57-H5H9HA: first, prepare the fusion gene H5H9HA, then add a kozak sequence to one end of the fusion gene H5H9HA sequence, then add an EcoRI restriction site, and add an NheI restriction site to the other end site; H5H9HA can be artificially synthesized with the full sequence, and the DNA sequences encoding the H5 HA antigen and the H9 HA antigen can also be obtained by PCR amplification, and then spliced ​​together to form the DNA sequence encoding the fusion protein of the present invention; or can be used For example, it is synthesized by overlapping PCR; then the pretreated fusion gene H5H9HA is cloned into the vector pUC57 to obtain the plasmid pUC57-H5H9HA with the target fusion gene H5H9HA;

[0042] S2, construction of the adenovirus shuttle plas...

Embodiment 1

[0057] Embodiment 1 of the present invention provides a method for preparing an adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes, comprising the following steps:

[0058] S1, preparation of plasmid pUC57-H5H9HA: (cloning of H5N6HA and H9N2HA fusion gene plasmid)

[0059] Screen the dominant strains of avian influenza H5N6 subtype and H9N2 subtype, and determine the HA gene sequence of the aforementioned strains, and accordingly synthesize the fusion gene H5N6HAH9N2HA (abbreviated as H5H9HA) of the protective antigen HA of H5N6 and H9N2, and then integrate the fusion gene H5H9HA is cloned into pUC57 vector, the above process is as follows:

[0060] 1. After removing the stop codon and subsequent sequences in the HA gene sequence of H5N6, immediately following the start codon of the HA gene sequence in H9N2, until the stop codon of the HA gene sequence in H9N2, the HA gene of H5N6 and H9N2 The HA gene is fused into a fusion gen...

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Abstract

The invention provides an adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes and a preparation method of the adenovirus vector bivalent vaccine. Hemagglutinin antigens of the avian influenza H5 subtype and H9 subtype are used as core antigens to synthesize a fusion gene H5H9HA, an adenovirus is used as a vector, the fusion gene H5H9HA is cloned to an adenovirus shuttle vector pDC315, then the fusion gene H5H9HA and skeleton plasmids pBHGlox (delta) E1 and 3Cre are co-transfected into 293 cells to complete recombinant adenovirus production; and then through amplification and purification, the high-purity recombinant adenovirus rAd-H5H9HA carrying the hemagglutinin antigens is obtained, and through freeze-drying and packaging post-treatment, an adenovirus vector bivalent vaccine capable of preventing avian influenza H5 and H9 subtypes at the same time is obtained. The vaccine can induce a high-level H5 subtype specific antibody and a high-level H9 subtype specific antibody, the level of the antibodies is consistent with that of an existing commercial totivirus inactivated vaccine and exceeds the immune standard specified by China, andthe vaccine has huge application prospects and popularization value in the field of avian influenza immunoprophylaxis.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to an adenovirus vector bivalent vaccine for simultaneously preventing avian influenza H5 and H9 subtypes and a preparation method thereof. Background technique [0002] Avian influenza is an acute, virulent, and contagious respiratory infectious disease of poultry caused by type A influenza virus. Avian influenza can be divided into highly pathogenic avian influenza and low pathogenic avian influenza. Highly pathogenic avian influenza has become an endemic disease in some Asian countries, and is listed as a "notifiable animal disease" by the World Organization for Animal Health (OIE), and is also listed as a first-class animal infectious disease in my country. Avian influenza can infect not only birds, but also humans and other mammals, so the prevention and control of avian influenza has public health significance. [0003] According to the difference of AIV hemagglut...

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Application Information

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IPC IPC(8): C12N15/62C12N15/861C12N15/66C07K19/00A61K39/145A61K39/295A61P31/16
CPCC07K14/005C12N15/86C12N15/66A61K39/12A61P31/16C12N2760/16122C07K2319/00C12N2760/16134C12N2710/10243C12N2800/106C12N2800/22A61K2039/53A61K2039/552A61K2039/70Y02A50/30
Inventor 王增利周立桥张哲李金华霍惠玲韩荞忆胡晓悦马贵达顾文源马宏伟韩庆安刘天驹仇国明甄理段英萍
Owner 河北省动物疫病预防控制中心
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