WO2019129254A1 - 一种流感病毒抗体的新应用 - Google Patents

一种流感病毒抗体的新应用 Download PDF

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WO2019129254A1
WO2019129254A1 PCT/CN2018/125134 CN2018125134W WO2019129254A1 WO 2019129254 A1 WO2019129254 A1 WO 2019129254A1 CN 2018125134 W CN2018125134 W CN 2018125134W WO 2019129254 A1 WO2019129254 A1 WO 2019129254A1
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influenza virus
seq
antibody
virus antibody
variable region
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PCT/CN2018/125134
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French (fr)
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高福
校海霞
郭天玲
陈维之
孙中平
洪媛媛
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中国科学院天津工业生物技术研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present application relates to the field of biotechnology, and relates to a novel application of an influenza virus antibody, and particularly to an application of an influenza virus antibody in vivo and an H7 subtype influenza virus.
  • Avian influenza is a type of zoonotic infectious disease caused by influenza A virus (IAV).
  • IAV influenza A virus
  • H subtypes H1-H16
  • N(N1-N9) subtypes of influenza A virus are present in waterfowl and seabirds, while only H17, H18 and N10, N11 subtypes exist in bats.
  • Waterfowl and seabirds are therefore known as the natural reservoir of influenza A virus.
  • Avian influenza can infect a variety of mammals, including humans, as well as a variety of wild and domestic poultry.
  • avian influenza can be divided into three categories: highly pathogenic avian influenza (HPAI), low pathogenic avian influenza (LPAI) and non-pathogenicity.
  • HPAI highly pathogenic avian influenza
  • LPAI low pathogenic avian influenza
  • NPAI non-pathogenic avian influenza
  • HPAI is a disease caused by H5 and H7 subtype influenza viruses represented by H5N1 and H7N7, which is a great hazard and seriously affects the development of the poultry industry.
  • H5N1 and H7N7 which is a great hazard and seriously affects the development of the poultry industry.
  • such viruses can cross the interspecific barriers and infect humans and therefore have important public health implications.
  • H7 subtype influenza virus is usually prevalent in poultry. Occasionally, some viruses such as H7N3, H7N2, and H7N7 are transmitted to humans across the species, but only cause some mild symptoms such as conjunctivitis and fever like common flu. There was a fatal respiratory distress in the case, but only one died. However, at the end of March 2013, there was an incident of low-pathogenic avian influenza H7N9 infection and death in Shanghai and Anhui, which is the first time in the world. Since then, the virus has expanded to Beijing, Jiangsu, Zhejiang, Shandong, Henan, Hunan, Jiangxi, Fujian and Taiwan. The H7N9 avian influenza virus did not stop in 2013.
  • H7N9 avian influenza From 2013 to 2017, there have been five outbreaks of H7N9 avian influenza, and the number and extent of infected poultry has increased, which has led to the use of the H7N9 vaccine for poultry. In the five rounds of epidemics, the number of H7N9 infections has also increased, and some H7N9 isolates have acquired the HA cleavage site characteristics of highly pathogenic avian influenza, becoming HAPIV, which is undoubtedly a public safety for humans. It constitutes a serious threat.
  • the drugs for clinical treatment of H7N9 influenza patients are mainly neuraminidase inhibitors.
  • the neuraminidase inhibitor is an antiviral drug targeting the neutrophil release process, targeting the neuraminidase (NA) of influenza virus.
  • NA neuraminidase
  • Neuraminidase inhibitors for the treatment of influenza including zanamivir and oseltamivir, have been shown to provide better therapeutic effects in patients with early influenza infection.
  • patients after treatment are susceptible to resistance to neuraminidase inhibitors, and some patients may develop resistance to them without treatment. Therefore, it is urgent to develop other novel therapeutic drugs specific for the H7N9 influenza virus.
  • the present application provides a new application of influenza virus antibodies, and develops a human antibody capable of neutralizing H7 subtype influenza virus in vivo, which will prevent and treat H7 subtype influenza.
  • Providing new alternative medicines has important economic and social implications.
  • the application provides an application of an influenza virus antibody for preparing a drug against an H7 subtype influenza virus
  • the light chain variable region of the influenza virus antibody has the amino acid sequence of SEQ ID NO. 1; and the heavy chain variable region of the influenza virus antibody has the amino acid sequence of SEQ ID NO.
  • amino acid sequence is as follows:
  • any one of the amino acid sequences substituted, deleted, added or introduced into one or two amino acids is also protected by the present application, which still has activity against the H7 subtype influenza virus.
  • the H7 subtype influenza virus includes H7N9 influenza virus, H7N1 influenza virus, H7N2 influenza virus, H7N3 influenza virus, H7N4 influenza virus, H7N5 influenza virus, H7N6 influenza virus, H7N7 influenza virus, H7N8 influenza virus or mutation thereof. Any one or a combination of at least two of the bodies.
  • the antibody prepared according to the present application is named AF4H1K1 antibody, and the key amino acid sequence of the corresponding H7 subtype HA protein recognized by it is relatively conservative.
  • AF4H1K1 recognizes and binds to the HA protein of the H7N9 influenza virus, and similarly recognizes other H7 subtype HA proteins.
  • the light chain antigen complementarity determining region of the influenza virus antibody has the amino acid sequence shown in any one of SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, and the influenza virus antibody
  • the heavy chain antigen complementarity determining region has the amino acid sequence set forth in any one of SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO.
  • amino acid sequence is as follows:
  • SEQ ID NO. 5 QSVSSSY;
  • SEQ ID NO. 7 QQYGSSFT
  • SEQ ID NO. 8 GFTSSAYA
  • SEQ ID NO. 10 ARDPLTKLLPFDWVSGGYFDY.
  • the present application provides a DNA fragment encoding an influenza virus antibody according to the application of the first aspect, which comprises the light chain variable region encoding the influenza virus antibody as set forth in SEQ ID NO.
  • the nucleotide sequence is as follows:
  • the application provides an expression vector comprising at least one copy of the DNA fragment of the second aspect.
  • the application provides a host cell, wherein the host cell comprises the expression vector of the third aspect.
  • the present application provides a medicament for anti-H7 subtype influenza virus, comprising the influenza virus antibody according to the application of the first aspect, the DNA fragment according to the second aspect, the third aspect Any one or a combination of at least two of the expression vector or the host cell of the fourth aspect.
  • the medicament further comprises a pharmaceutically acceptable carrier.
  • the present invention provides a method for preparing a medicament according to the fifth aspect, comprising the steps of:
  • PBMC peripheral blood mononuclear cells
  • the synthesized antibody fragment is constructed into an expression vector.
  • the vector of step (3) is a mammalian expression vector.
  • influenza virus antibody described in the present application has a good neutralizing effect on the highly pathogenic H7N9 subtype influenza virus in vivo, and the antibody can prevent the highly pathogenic H7N9 subtype influenza virus infection from lethal mice;
  • the antibody described in the present application can treat mice infected with the H7 subtype influenza virus, and has a 100% cure rate for mice infected with the highly pathogenic H7N9 influenza virus;
  • FIG. 2 is an evaluation of the prophylactic efficacy of the antibody AF4H1K1 prepared in the present application in BALB/c mice, wherein FIG. 2(a) is a change in body weight of the mouse, and FIG. 2(b) is a survival rate of the mouse;
  • FIG. 3 is an evaluation of therapeutic efficacy of the antibody AF4H1K1 prepared in the present application in BALB/c mice, wherein FIG. 3(a) is a change in body weight of the mouse, and FIG. 3(b) is a survival rate of the mouse;
  • Figure 4 is a graphical representation of the alignment of the antibody AF4H1K1 prepared in the present application to recognize key amino acid positions of HA proteins such as H3 and H7 subtypes.
  • the preparation method of the influenza virus antibody comprises the following steps:
  • the nucleotide sequence of the VL is:
  • the nucleotide sequence of the VH is:
  • the antibody IgG fragment is constructed into a mammalian expression vector
  • a mammalian expression vector into which a heavy chain and a light chain sequence were inserted was co-transfected into 293T cells by PEI transfection reagent for large-scale expression and purified.
  • the antibody AF4H1K1 was injected into the tail vein of mice (6 weeks old BALB/c, female) at a dose of 15 mg/kg, and the Ebola virus neutralizing antibody 13C6 (human-mouse hybrid antibody) was set as a negative control.
  • the mice were anesthetized with dry ice after 24 hours of antibody injection, and then intranasally infected with highly pathogenic H7N9 influenza virus of 10 7 EID 50 .
  • Five rats in each group were observed for death after the challenge, and the body weight of the mice was weighed until the 14th day after the challenge. The results are shown in Fig. 2(a) - Fig. 2(b).
  • Fig. 2(a) It can be seen from Fig. 2(a) that in the AF4H1K1 prevention group, the body weight of the mice gradually decreased with the increase of the feeding days, and it can be seen that the AF4H1K1 prevention group could not inhibit the weight loss of the mice, but from Fig. 2(b) It can be seen that in the AF4H1K1 prevention group, the death of the mice can be 100% prevented by injecting the antibody through the tail vein 24 hours before the challenge. The mice in the negative control 13C6 antibody injection group were 100% mortality.
  • the AF4H1K1 antibody has a 100% preventive effect on mice infected with high dose H7N9 virus.
  • the highly pathogenic H7N9 influenza virus infected with 10 7 EID 50 was intranasally injected into the tail vein of the antibody AF4H1K1 at a dose of 15 mg/kg 24 h after infection, and the Ebola virus neutralizing antibody 13C6 was set. (Human-murine hybrid antibody) was a negative control. On the 0th to 14th day after the challenge, the body weight of the mice was weighed, and the survival rate of the mice was recorded. The results are shown in Fig. 3 (a) - Fig. 3 (b).
  • the AF4H1K1 antibody has a 100% therapeutic effect on mice infected with high dose H7N9 virus.
  • AF4H1K1 recognizes and binds to the HA protein of the H7N9 influenza virus, and determines that the AF4H1K1 recognizes the epitope HA sequence of the H7 subtype influenza virus.
  • various H7 subtypes H7N1, H7N2 entered in NCBI will be entered.
  • the HA sequences of H7N3, H7N4, H7N5, H7N6, H7N7 and H7N8 were sequence-aligned with the HA protein of H7N9 influenza virus, and the results are shown in FIG.
  • AF4H1K1 can bind H7N1, H7N2, H7N3 in addition to H7N9 subtype influenza virus.
  • Other subtypes of influenza viruses such as H7N4, H7N5, H7N6, H7N7 and H7N8.
  • influenza virus antibody described in the present application can well neutralize the influenza virus of the H7 subtype, and particularly has a good neutralizing effect on the highly pathogenic H7N9 influenza virus, and the antibody can prevent high incidence.
  • the diseased H7N9 subtype influenza virus infects dead mice.

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Abstract

提供了一种流感病毒抗体的新应用，具体地为一种流感病毒抗体用于制备抗H7亚型流感病毒药物的应用，其中，所述流感病毒抗体的轻链可变区具有SEQ ID NO.1所示的氨基酸序列；所述流感病毒抗体的重链可变区具有SEQ ID NO.2所示的氨基酸序列。所述流感病毒抗体能够很好的结合H7亚型的流感病毒的HA蛋白，尤其针对H7N9流感病毒在体内具有很好的中和效果，该抗体能预防H7N9亚型流感病毒感染致死小鼠。

Description

一种流感病毒抗体的新应用 技术领域
本申请涉及生物技术领域，涉及一种流感病毒抗体的新应用，具体涉及一种流感病毒抗体在体内中和H7亚型流感病毒的应用。
背景技术
禽流感(avian influenza，AI)是由A型流感病毒(influenza A virus，IAV)引起的一类人畜共患传染性疾病。研究发现，A型流感病毒的H亚型(H1-H16)和N(N1-N9)亚型都存在于水禽及海鸟中，而只有H17、H18和N10、N11亚型病毒存在于蝙蝠这一哺乳动物体内，目前还没有感染水禽的报道。因此水禽和海鸟被称为A型流感病毒的天然存贮库(natural reservoir)。禽流感可感染包括人在内的多种哺乳动物及多种野生及家养禽类。根据致病性的不同，禽流感可分为三大类：高致病性禽流感(highly pathogenic avian influenza，HPAI)、低致病性禽流感(low pathogenic avian influenza，LPAI)和非致病性禽流感(non-pathogenic avian influenza，NPAI)。其中HPAI是以H5N1和H7N7为代表的H5和H7亚型流感病毒引起的疾病，危害巨大，严重影响了养禽业的发展。此外，这类病毒可跨越种间障碍，进而感染人类，因此具有重要的公共卫生意义。
H7亚型流感病毒通常在禽类中流行，偶尔会有一些病毒如H7N3、H7N2、和H7N7跨种间传播到人，只是引起一些轻微的症状，如结膜炎和类似普通流感的发烧等症状，个别病例出现致命的呼吸窘迫症，但是只有1例死亡。然而，在2013年3月底在上海和安徽出现了低致病性禽流感H7N9感染并致死人的事件，这在全球尚属首次。此后，该病毒扩展到了了北京、江苏、浙江、山 东、河南、湖南、江西、福建和台湾等地区。H7N9禽流感病毒并没有止步于2013年。自2013年至2017年，出现了五轮H7N9禽流感的暴发，感染家禽的数量和范围也随着加大，由此催生了禽用H7N9疫苗的使用。在这五轮的流行中，H7N9感染人数也随之增多，且有些H7N9分离毒株已获得了高致病性禽流感的HA裂解位点特征，成为了HAPIV，这无疑对人类的公共安全却构成了一严重的威胁。
目前H7N9流感患者临床治疗用的药物主要是神经氨酸酶抑制剂。神经氨酸酶抑制剂是以流感病毒的神经氨酸酶(neuraminidase，NA)为靶标，针对病毒粒子释放过程的抗病毒药物。市场上已有治疗流感的神经氨酸酶抑制剂包括扎那米韦和奥司他韦，对感染流感早期病人提供较好的治疗效果。然而治疗后的病人容易对神经氨酸酶抑制剂产生耐药性，有些病人在没有使用治疗的情况下也会对其产生耐药性。因此亟需研制出其他新型的H7N9流感病毒特异的治疗性药物。
发明内容
针对现有技术的不足及实际的需求，本申请提供一种流感病毒抗体的新应用，研制出了可以体内中和H7亚型流感病毒的人源抗体，将为H7亚型流感的预防和治疗提供新的备选药物，具有重要的经济和社会意义。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种流感病毒抗体用于制备抗H7亚型流感病毒的药物的应用；
其中，所述流感病毒抗体的轻链可变区具有SEQ ID NO.1所示的氨基酸序列；所述流感病毒抗体的重链可变区具有SEQ ID NO.2所示的氨基酸序列。
所述的氨基酸序列如下：
SEQ ID NO.1：
Figure PCTCN2018125134-appb-000001
SEQ ID NO.2：
Figure PCTCN2018125134-appb-000002
本申请中，所述氨基酸序列中，取代、缺失、添加或导入1-2个氨基酸的任意一种氨基酸序列也在本申请的保护中，其仍然具有抗H7亚型流感病毒的活性。
根据本申请，所述H7亚型流感病毒包括H7N9流感病毒、H7N1流感病毒、H7N2流感病毒、H7N3流感病毒、H7N4流感病毒、H7N5流感病毒、H7N6流感病毒、H7N7流感病毒、H7N8流感病毒或其突变体中的任意一种或至少两种的组合。
根据本申请制备的抗体被命名为AF4H1K1抗体，它所识别的对应的H7亚型HA蛋白的关键氨基酸序列是相对保守的。AF4H1K1可以识别并结合H7N9流感病毒的HA蛋白，同理也可识别其他H7亚型HA蛋白。
根据本申请，所述流感病毒抗体的轻链抗原互补决定区具有SEQ ID NO.5、SEQ ID NO.6和SEQ ID NO.7中任意一种所示的氨基酸序列；所述流感病毒抗体的重链抗原互补决定区具有SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10中任意一种所示的氨基酸序列。
所述的氨基酸序列如下：
SEQ ID NO.5：QSVSSSY；
SEQ ID NO.6：RAS；
SEQ ID NO.7：QQYGSSFT；
SEQ ID NO.8：GFTSSAYA；
SEQ ID NO.9：ITFDGGYQ；
SEQ ID NO.10：ARDPLTKLLPFDWVSGGYFDY.
第二方面，本申请提供一种编码如第一方面所述应用中的流感病毒抗体的DNA片段，其包含如SEQ ID NO.3所示的编码所述流感病毒抗体的轻链可变区的核苷酸序列，以及如SEQ ID NO.4所示的编码所述流感病毒抗体的重链可变区的核苷酸序列。
所述的核苷酸序列如下：
SEQ ID NO.3：
Figure PCTCN2018125134-appb-000003
SEQ ID NO.4：
Figure PCTCN2018125134-appb-000004
第三方面，本申请提供一种表达载体，所述表达载体包含至少一个拷贝的如第二方面所述的DNA片段。
第四方面，本申请提供一种宿主细胞，其中，所述宿主细胞包含如第三方面所述的表达载体。
第五方面，本申请提供一种抗H7亚型流感病毒的药物，所述药物包括如第一方面所述的应用中的流感病毒抗体、如第二方面所述的DNA片段、如第三方面所述的表达载体或如第四方面所述的宿主细胞中的任意一种或至少两种的组合。
根据本申请，所述药物还包括药学上可接受的载体。
第六方面，本申请提供一种如第五方面所述的药物的制备方法，包括如下步骤：
(1)分离感染者外周血单个核细胞(peripheral blood mononuclear cell，PBMC)，提取RNA，反转录cDNA；
(2)扩增重链和轻链的高度可变区序列，根据互补决定区(complementarity determining region，CDR)丰度进行挑选并合成；
(3)将合成的抗体片段构建到表达载体中。
优选地，步骤(3)所述的载体为哺乳动物表达载体。
与现有技术相比，本申请具有如下有益效果：
(1)本申请所述流感病毒抗体对高致病性H7N9亚型流感病毒在体内具有很好的中和效果，该抗体能预防高致病性H7N9亚型流感病毒感染致死小鼠；
(2)本申请所述抗体能治疗H7亚型流感病毒感染的小鼠，针对高致病性H7N9流感病毒感染的小鼠具有100％的治愈率；
(3)本申请所述抗体的获得为流感的预防和治疗提供了新的途径，具有重要的经济和社会意义。
附图说明
图1是本申请制备的抗体IgG和Fab经SuperdexTM200 10/300 GL分子筛层析纯化结果；
图2是本申请制备的抗体AF4H1K1在BALB/c小鼠体内预防效力评估，其中，图2(a)为小鼠体重变化，图2(b)为小鼠存活率；
图3是本申请制备的抗体AF4H1K1在BALB/c小鼠体内治疗效力评估，其中，图3(a)为小鼠体重变化，图3(b)为小鼠存活率；
图4是本申请制备的抗体AF4H1K1在识别H3和H7亚型等HA蛋白的关键氨基酸位点的比对分析图。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1抗体的制备和纯化
所述的流感病毒抗体的制备方法，包括如下步骤：
(1)分离感染者外周血中的PBMC，提取RNA，反转录cDNA；
(2)扩增出重链和轻链的高度可变区序列，将扩增的目的片段利用Miseq 2X 300bp进行测序，并对测序结果进行分析；
(3)以CDR丰度为主要参数挑选感染患者的高频可变区序列，并通过重链轻链配对算法计算自然配对的几率，接着挑选出CDR1、CDR2和CDR3的高频出现的VH和VL序列加上各自的恒定区进行合成；
所述VL的核苷酸序列为：
Figure PCTCN2018125134-appb-000005
所述VH的核苷酸序列为：
Figure PCTCN2018125134-appb-000006
(4)在N端加入对应的重链和轻链的分泌信号肽后，将抗体IgG片段构建到哺乳动物表达载体；
(5)将插入重链和轻链序列的哺乳动物表达载体，通过PEI转染试剂共转染293T细胞进行大量表达，并进行纯化。
(6)纯化具体步骤：在转染后96h后收获上清，5000rpm，离心1h，上清经过0.22μM的滤膜过滤，蠕动泵过夜将上清结合Protein A预装柱，AKTA机器上用0.1M的甘氨酸洗脱结合在Protein A上面的IgG蛋白。洗脱的蛋白浓缩换buffer(10mM Tris，40mM NaCl)后，经过Superdex TM200 10/300 GL分子筛进一步纯化，纯化后的抗体命名为AF4H1K1。
纯化结果如图1所示，可见，通过纯化后都能得到单一所要抗体的IgG。
实施例2 H7N9流感病毒体内抗体预防效果
(1)预防实验
通过小鼠(6周龄BALB/c，雌性)尾静脉注射抗体AF4H1K1，剂量为15mg/kg，同时设定埃博拉病毒中和抗体13C6(人-鼠杂合抗体)为阴性对照。抗体注射24h后干冰麻醉小鼠，而后滴鼻感染10 7 EID 50的高致病性H7N9流感病毒。每组5只，攻毒后，观察小鼠的死亡，并称取小鼠的体重，直至攻毒后第14天，结果如图2(a)-图2(b)所示。
从图2(a)可以看出，AF4H1K1预防组中，小鼠的体重随着饲养天数的增加呈逐渐下降的趋势，可见AF4H1K1预防组不能抑制小鼠体重的下降，但是从图2(b)可以看出，AF4H1K1预防组中，在攻毒前24小时通过尾静脉注射抗体后，可以100％预防小鼠的死亡。而阴性对照13C6抗体注射组的小鼠却是100％的死亡率。
可见，根据小鼠预防实验可以看出AF4H1K1抗体对高剂量H7N9病毒感染的小鼠有100％的预防效果。
(2)治疗实验
首先，滴鼻感染10 7 EID 50的高致病性H7N9流感病毒，在感染后24h，通过小鼠尾静脉注射抗体AF4H1K1，剂量分别为15mg/kg，同时设定埃博拉病毒中和抗体13C6(人-鼠杂合抗体)为阴性对照。攻毒后第0-第14天，称取小鼠的体重，记录小鼠存活率，结果如图3(a)-图3(b)所示。
结果发现，从图3(a)可以看出，AF4H1K1治疗组不能抑制小鼠体重的下降，但是从图3(b)可以看出，AF4H1K1治疗组中，在攻毒后24小时通过尾静脉注射抗体后，可以对小鼠提供100％保护率，而阴性对照13C6抗体注射组的小鼠却是100％的死亡率。
可见，根据小鼠治疗实验可以看出AF4H1K1抗体对高剂量H7N9病毒感染的小鼠有100％的治疗效果。
实施例3 AF4H1K1对其他H7亚型的流感病毒的保护效果
本申请中，AF4H1K1可以识别并结合H7N9流感病毒的HA蛋白，确定了AF4H1K1识别H7亚型流感病毒的抗原表位HA序列，为此，将录入在NCBI中的各种H7亚型(H7N1、H7N2、H7N3、H7N4、H7N5、H7N6、H7N7和H7N8)的HA序列与H7N9流感病毒的HA蛋白进行序列比对分析，结果如图4所示。
从图4可以看出AF4H1K1抗体所识别的对应的H7亚型HA蛋白的关键氨基酸序列是相对保守的，显然，AF4H1K1除结合和识别H7N9亚型流感病毒外，还可以结合H7N1、H7N2、H7N3、H7N4、H7N5、H7N6、H7N7和H7N8等其他亚型流感病毒。
综上所述，本申请所述流感病毒抗体能够很好的中和H7亚型的流感病毒，尤其针对高致病性H7N9流感病毒在体内具有很好的中和效果，该抗体能预防高致病性H7N9亚型流感病毒感染致死小鼠。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (11)

  1. 一种流感病毒抗体用于制备抗H7亚型流感病毒的药物的应用；
    其中，所述流感病毒抗体的轻链可变区具有SEQ ID NO.1所示的氨基酸序列；所述流感病毒抗体的重链可变区具有SEQ ID NO.2所示的氨基酸序列。
  2. 根据权利要求1所述的应用，其中，所述H7亚型流感病毒包括H7N9流感病毒、H7N1流感病毒、H7N2流感病毒、H7N3流感病毒、H7N4流感病毒、H7N5流感病毒、H7N6流感病毒、H7N7流感病毒、H7N8流感病毒或其突变体中的任意一种或至少两种的组合。
  3. 根据权利要求1或2所述的应用，其中，所述流感病毒抗体的轻链抗原互补决定区具有SEQ ID NO.5、SEQ ID NO.6和SEQ ID NO.7中任意一种所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的应用，其中，所述流感病毒抗体的重链抗原互补决定区具有SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10中任意一种所示的氨基酸序列。
  5. 一种编码如权利要求1-4中任一项所述应用中的流感病毒抗体的DNA片段，其包含如SEQ ID NO.3所示的编码所述流感病毒抗体的轻链可变区的核苷酸序列，以及如SEQ ID NO.4所示的编码所述流感病毒抗体的重链可变区的核苷酸序列。
  6. 一种表达载体，其包含至少一个拷贝的如权利要求5所述的DNA片段。
  7. 一种宿主细胞，其包含权利要求6所述的表达载体。
  8. 一种抗H7亚型流感病毒的药物，其包括如权利要求1-3中任一项所述的应用中的流感病毒抗体、如权利要求4所述的DNA片段、如权利要求5所述的表达载体或如权利要求6所述的宿主细胞中的任意一种或至少两种的组合。
  9. 根据权利要求8所述的药物，其中，所述药物还包括药学上可接受的载体。
  10. 一种如权利要求8或9所述的药物的制备方法，其包括如下步骤：
    (1)分离感染者外周血中的PBMC，提取RNA，反转录cDNA；
    (2)扩增重链和轻链的高度可变区序列，根据CDR丰度进行挑选并合成；以及
    (3)将合成的抗体片段构建到表达载体中。
  11. 根据权利要求10所述的制备方法，其中，步骤(3)所述的载体为哺乳动物表达载体。
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