WO2023066396A1 - 靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组及应用 - Google Patents

靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组及应用 Download PDF

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WO2023066396A1
WO2023066396A1 PCT/CN2022/126859 CN2022126859W WO2023066396A1 WO 2023066396 A1 WO2023066396 A1 WO 2023066396A1 CN 2022126859 W CN2022126859 W CN 2022126859W WO 2023066396 A1 WO2023066396 A1 WO 2023066396A1
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amino acid
acid sequence
igy
pabs
new coronavirus
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French (fr)
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黑爱莲
李劲
何艾伦
斯库格斯文
周际
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华瑞同康生物技术（深圳）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • This application relates to the field of biomedicine, more specifically, it relates to a core amino acid sequence group and its application for targeting recognition of anti-new coronavirus neutralizing antibody N-IgY-pAbs (neutralizing IgY polyclonal antibodies).
  • novel coronavirus pneumonia (COVID-19 for short) epidemic is an acute infectious disease event that is the most concerned in the world today. Since the epidemic broke out in the world, it has not been effectively and completely contained. Novel coronavirus (new coronavirus for short) has infected a large number of people, is highly contagious, and has a high fatality rate. So far, great progress has been made in the development and treatment of COVID-19 vaccines. Vaccines have been promoted and marketed, but there is still no specific drug for the treatment of COVID-19. Therefore, there is still an urgent need to explore new methods, new means, and new tools for the prevention, diagnosis and treatment of the novel coronavirus pneumonia epidemic.
  • the use of new coronavirus-specific antibodies can neutralize the virus, thereby preventing the virus from adhering to and invading host cells.
  • the new coronavirus infection body first binds the receptor binding domain (RBD) on the spike protein (S protein) to the receptor angiotensin-converting enzyme 2 (ACE2) of the host cell, and mediates the entry of the virus into the host cell. Therefore, in terms of immunogen selection, the use of the new coronavirus S protein extracellular domain (S-ECD) is an ideal and effective immunization antigen, which can effectively induce the production of neutralizing antibodies.
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • Anti-new coronavirus antibodies can generally be divided into two categories, namely neutralizing antibodies (NAbs, Neutralizing Antibodies) and non-neutralizing virus-binding antibodies (BAbs, Binding Antibodies).
  • NAbs neutralizing antibodies
  • BAbs Binding Antibodies
  • the new coronavirus S protein is a key target of NAbs, because the new coronavirus invades the host cell through the interaction between its S protein and the host cell surface ACE2 protein.
  • BAbs can bind to all protein components of the new coronavirus, including S, N, E and M proteins.
  • NAbs are one of the most important criteria for predicting the success of a COVID-19 vaccine.
  • the new coronavirus has mutated at different speeds.
  • researchers have analyzed the mutation rates of 27 different proteins at different times and found that the mutation rates of different proteins of the new coronavirus are quite different.
  • the S protein and N protein in the new coronavirus vaccine and treatment have the highest mutation variability, such as D614G (S protein), P323L (NSP12) and R203K/G204R (N protein). More recent mutations have identified other sites such as A222V (S protein), L18F (S protein), and A220V (N protein) (Vilar S, Isom D G. One Year of SARS-CoV-2: How Much Has the Virus Changed?. Biology, 2021, 10:91).
  • S protein S protein
  • T478K L452R
  • P681R E484Q
  • the inventor believes that in the evaluation of antibody-specific binding target sequence, whether there is a mutation region and whether it contains a mutation point is an important indicator for performance evaluation. It is of great significance and application value to discover the conserved amino acid sequence that binds to specific antibodies, especially neutralizing antibodies.
  • this application provides a core amino acid sequence group and its application for the targeted recognition of anti-new coronavirus neutralizing antibody N-IgY-pAbs.
  • the core amino acid sequence group of a targeted recognition anti-new coronavirus neutralizing antibody N-IgY-pAbs provided by the application adopts the following technical scheme:
  • a core amino acid sequence group that targets and recognizes anti-new coronavirus neutralizing antibody N-IgY-pAbs including the following amino acid sequences:
  • amino acid sequence located in the S-ECD region is numbered according to the protein sequence as follows:
  • S-NTD 21 RTQLPPAYTNSFTRG 35 , 141 LGVYYHKNNKSWMES 155 , 261 GAAAYYVGYLQPRTF 275 and 291 CALDPLSETKCTLKS 305 ;
  • S-RBD 411 APGQTGKIADYNYKL 425 and 461 LKPFERDISTEIYQA 475 ;
  • S-CTD1 561 PFQQFGRDIADTTDA 575 , 571 DTTDAVRDPQTLEIL 585 and 581 TLEILDITPCSFGGV 595 ;
  • S1/S2 junction area 741 YICGDSTECSSNLLLQ 755 , 811 KPSKRSFIEDLLFNK 825 and 821 LLFNKVTLADAGFIK 835 ;
  • amino acid sequence located in the nonstructural protein region is numbered according to the protein sequence as follows:
  • ORF1ab 1361 SNEKQEILGTVSWNL 1375 ;
  • ORF1ab 6411 HHANEYRLYLDAYNM 6425 ;
  • ORF10 21 MNSRNYIAQVDVVNFNLT 38 ;
  • ORF7a 1 MKIILFLALITLATC 15 and 111 TLCFTLKRKTE 121 .
  • the core amino acid sequence group of the targeted recognition anti-new coronavirus neutralizing antibody N-IgY-pAbs consists of the following amino acid sequences:
  • amino acid sequence located in the S-ECD region is numbered according to the protein sequence as follows:
  • S-NTD 21 RTQLPPAYTNSFTRG 35 , 141 LGVYYHKNNKSWMES 155 , 261 GAAAYYVGYLQPRTF 275 and 291 CALDPLSETKCTLKS 305 ;
  • S-RBD 411 APGQTGKIADYNYKL 425 and 461 LKPFERDISTEIYQA 475 ;
  • S-CTD1 561 PFQQFGRDIADTTDA 575 , 571 DTTDAVRDPQTLEIL 585 and 581 TLEILDITPCSFGGV 595 ;
  • S1/S2 junction area 741 YICGDSTECSSNLLLQ 755 , 811 KPSKRSFIEDLLFNK 825 and 821 LLFNKVTLADAGFIK 835 ;
  • amino acid sequence located in the nonstructural protein region is numbered according to the protein sequence as follows:
  • ORF1ab 1361 SNEKQEILGTVSWNL 1375 ;
  • ORF1ab 6411 HHANEYRLYLDAYNM 6425 ;
  • ORF10 21 MNSRNYIAQVDVVNFNLT 38 ;
  • ORF7a 1 MKIILFLALITLATC 15 and 111 TLCFTLKRKTE 121 .
  • MRs 0.01–0.025
  • MRs 0.025-0.05
  • target amino acid sequences recognized by the above 20 high-efficiency neutralizing antibodies 15 were identified to be located in the S-ECD region, and 5 were identified to be located in the nonstructural protein (NSP) region.
  • the amino acid sequence is a correspondingly adjusted or modified amino acid sequence
  • the modified materials include one or more of nanomaterials, fluorescent materials, enzymes, biotin and proteins.
  • the modified materials include but are not limited to nanomaterials, fluorescent materials, enzymes, biotin and specific proteins, which are beneficial to Apply the adjusted or modified amino acid sequence to the detection of the new coronavirus, the immune antigen design of the new coronavirus vaccine, and the evaluation of the new coronavirus vaccine.
  • the application provides an application for targeting the core amino acid sequence group of anti-new coronavirus neutralizing antibody N-IgY-pAbs, adopting the following technical scheme:
  • An application of the core amino acid sequence group targeting recognition of anti-new coronavirus neutralizing antibody N-IgY-pAbs with one or more of each amino acid sequence as the core, applied to the detection of new coronavirus, or used in the preparation of detection Reagents or kits for novel coronavirus.
  • the above-mentioned amino acid sequences have high specificity and high affinity characteristics, and can be effectively applied to the quantitative and/or qualitative detection of new coronaviruses by using one or more of each amino acid sequence as the core.
  • the detection includes but not limited to ELISA detection, immunochemiluminescence detection and immunofluorescence detection.
  • each amino acid sequence can be used in various detection methods such as ELISA detection, immunochemiluminescence detection and immunofluorescence detection, and has a wide range of applications and strong applicability.
  • the above-mentioned amino acid sequences have high specificity and high affinity characteristics. Taking one or more of each amino acid sequence as the core and applying them to the design of new coronavirus therapeutic targets, it is possible to design a therapeutic preparations.
  • the therapeutic target design includes but is not limited to the target design of therapeutic antibodies and the target design of non-antibody therapeutic drugs.
  • each amino acid sequence can be applied to target design of therapeutic antibodies and non-antibody therapeutic drugs, with wide application range and strong applicability.
  • the above-mentioned amino acid sequences have high specificity and high affinity characteristics, and one or more of each amino acid sequence can be applied to the target design of new coronavirus vaccines, effectively used to deal with the current new coronavirus The adverse situation of high-frequency mutations.
  • the vaccine target design includes but not limited to vaccine immune antigen design and vaccine performance evaluation.
  • each amino acid sequence can be applied to the design of vaccine immune antigens and the evaluation of vaccine performance, with wide application range and strong applicability.
  • Figure 1 is a detection map of the new coronavirus proteome chip in the embodiment of the present application, detecting the immune response of the anti-new coronavirus neutralizing antibody N-IgY-pAbs recognizing different polypeptide binding sites, N-IgY-pAbs (375ng/ml).
  • Figure 2 is a detection diagram of the new coronavirus proteome chip in the embodiment of the present application, detecting the immune response of unimmunized hen serum to recognize different polypeptide binding sites, unimmunized hen serum (1:2000).
  • Figure 3 is the detection map of the new coronavirus proteome chip in the embodiment of the present application.
  • the distribution map of the immune response is obtained according to the Z score > 0.05.
  • the Z score > 3.0 is considered as a significant strong signal, and the Z score ⁇ 5.0 is considered as Significant peak signal.
  • FIG. 4 is a schematic diagram of the S-ECD protein sequence (1273 amino acid residues).
  • the S-ECD in the figure shows: two main domains S1 (head) and S2 (stem region). Different structural domains are marked with different colors, and the primary structural sequence distribution names: SP (initial end), NTD (N-terminal domain); RBD (receptor binding domain) includes RBM (receptor binding motif), CTD1 (C-terminal domain 1), CTD2 (C-terminal domain 2), S1/S2 boundary (S1/S2 junction), S2' (S2' cleavage site), FL (fusion loop), FPPR ( Fusion peptide proximal region), HR1 (heptad repeat 1), CE (central helix), CD (linker domain), HR2 (heptad repeat 2), TM (transmembrane domain) and CT ( C terminal).
  • SP initial end
  • NTD N-terminal domain
  • RBD receptor binding domain
  • CTD1 C-termin
  • Figure 5 shows the amino acid sequences with a Z score > 0.05 in other structural domains except S-ECD, among which there are 5 amino acid sequences in the non-structural protein domain, and a Z score > 3.0 is considered as a significant strong signal.
  • the preparation method of the IgY neutralizing antibody against the new coronavirus proposed by the Chinese invention patent with the notification number CN112094341B is used to immunize the hen with the extracellular domain of the spike protein of the new coronavirus (S-ECD protein), extract the egg yolk antibody, and screen And preparation of anti-new coronavirus neutralizing antibody N-IgY-pAbs.
  • Hybridization reaction of N-IgY-pAbs the proteome chip was placed in a culture dish, and was mixed with PBST containing 5% (w/v) milk and 0.2% (v/v) Tween-20 at room temperature Block for 10 minutes. After washing, microarrays 1 and 2 were incubated with N-IgY-pAbs, respectively (N-IgY-pAbs concentrations were 375 ng/mL and 186 ng/mL, respectively). At the same time, pre-immune hen serum was added to microarray 3 (serum diluted 2000x) and microarray 4 (serum diluted 4000x), both incubated for 30 minutes. Subsequently, after washing three times, the chip was incubated with goat anti-chicken secondary antibody (Abcam, USA) labeled with Alexa Fluor 555 for 20 minutes at room temperature. Then, dry with a vacuum pump.
  • Abcam goat anti-chicken secondary antibody
  • the median fluorescence signal intensity of each spot was extracted using GenePix Pro7 software (Molecular Devices, USA).
  • the raw fluorescence signal intensity was the median signal intensity of each spot minus the median background intensity of each spot, and then the average value of duplicate wells was calculated.
  • the resulting signal was normalized by Z-score. The higher the Z score, the stronger the reaction signal, the more specific the recognition of the anti-new coronavirus neutralizing antibody N-IgY-pAbs, and the stronger the affinity.
  • a Z-score >3.0 was considered a significant strong signal, while a Z-score ⁇ 5.0 was considered a significant peak signal.
  • the results showed that the 4 negative controls in the control group had no signal, and only the mixture of IgG and IgM in the 2 positive controls showed strong positive, but the human poliovirus polypeptide line had no signal, indicating that the anti-new coronavirus neutralizing antibody N-IgY -pAbs does not recognize human poliovirus polypeptides, because human poliovirus polypeptides have nothing to do with the new coronavirus, so there is no signal display.
  • the anti-new coronavirus neutralizing antibody N-IgY-pAbs can significantly recognize S1+S2, S1 and S2 protein positive quality controls, reaching a significant peak signal (Z score) ⁇ 5.0, which verifies the anti-new coronavirus neutralizing antibody N-IgY - High sensitivity and high specificity of pAbs.
  • the reaction signals of the 20 amino acid sequences with a Z score greater than 3.0 on the proteome chip are shown in Table 2.
  • Table 2 the reaction signals of the 20 amino acid sequences with a Z score greater than 3.0 on the proteome chip are shown in Table 2.
  • only one amino acid sequence contains a S protein low frequency mutation site (MRs ⁇ 0.025 , S-NTD protein P272), and the remaining 19 amino acid sequences are highly conserved sequences, which do not contain the virus mutation sites found so far, and can be used for the design, development and research of novel coronavirus responses against high mutation frequencies.
  • amino acid sequences with high-efficiency neutralizing effects 15 were identified to be located in the S-ECD region, and 5 were identified to be located in the nonstructural protein (NSP) region.
  • the amino acid sequences are shown in Table 2 for details.
  • Vero cells (10 5 /mL) into a 96-well plate, 100 ⁇ l per well, and culture overnight at 37° C. in a CO 2 incubator. When the cell density reaches 80%-90%, wash and set aside.
  • step 3 Take the spare Vero cells in the 96-well plate, discard the supernatant, add 100 ⁇ L of the antibody and virus mixture of each antibody concentration in step 2) into the cells, and then add 100 ⁇ L of maintenance medium (DMEM containing 2% fetal bovine serum for culture) Base), cultured at 37°C for 48 hours.
  • maintenance medium DMEM containing 2% fetal bovine serum for culture
  • the preparation components include anti-new coronavirus neutralizing antibody N-IgY-pAbs; sodium chloride or mannitol; sterile deionized water or water for injection.
  • the content of anti-new coronavirus neutralizing antibody N-IgY-pAbs is 3-15 ⁇ g/ml, when adding sodium chloride, the mass concentration of sodium chloride is 0.9%; when adding mannitol, the content of mannitol is 10-30g/L .
  • Step 1 Filter the anti-new coronavirus neutralizing antibody N-IgY-pAbs through a 0.22 ⁇ m filter membrane.
  • Step 2 Take 10 ⁇ g of the anti-new coronavirus neutralizing antibody N-IgY-pAbs solution, add sterile deionized water, and make the anti-new coronavirus neutralizing antibody N-IgY-pAbs solution and sterile deionized water at a ratio of 1:3 Dilute proportionally, add sodium chloride to adjust the final concentration to 0.9%, and adjust the pH value to 7.4.
  • Step 3 filter with a 0.22 ⁇ m vacuum filter bottle, and pack in sterile sub-package bottles.
  • Antigen coating Dilute the antigen S protein RBD or S-ECD with coating solution to a concentration of 0.5ug/mL, 100uL per well, and coat overnight at 4°C;
  • Blocking after washing the plate with the discarded solution add 200 uL/well of blocking solution, and block for 1 hour at 37°C.
  • biotinylated secondary antibody add 250-fold diluted biotinylated secondary antibody 100uL/well, react at 37°C for 1 hour;
  • the stability data of the anti-new coronavirus neutralizing antibody N-IgY-pAbs spray formulation is shown in Table 4. The results show that the anti-new coronavirus neutralizing antibody N-IgY-pAbs spray formulation can be stored stably for at least 12 months at 4°C. There was no significant change in OD value (p>0.05).
  • the anti-new coronavirus neutralizing antibody N-IgY-pAbs spray preparation prepared in application example 2 has a virus inhibition rate of more than 99% for the new coronavirus Omicron virus strain.
  • the identification method comprises the steps of: detecting the neutralization effect of the spray preparation on the in vitro infection of Vero E6 cells by the new coronavirus variant Omicron variant by the focus reduction neutralization test (FRNT) method. The results showed that at a concentration of 305 ⁇ g/ml, the average neutralizing activity was 99.53%.
  • the anti-new coronavirus neutralizing antibody N-IgY-pAbs spray preparation prepared in Example 2 was used to quantitatively determine N by N gene and ORF1ab gene by RT-PCR method.
  • the results of the whole proteome chip test showed that the anti-new coronavirus N-IgY-pAbs has the characteristics of specifically recognizing a variety of target amino acid sequences, and these target amino acid sequences can cooperate with each other to promote the combination of anti-new coronavirus N-IgY-pAbs and
  • the virus receptor binding domain (RBD) interacts and blocks the binding of RBD to the host receptor angiotensin-converting enzyme-2 (ACE2), thus effectively neutralizing the novel coronavirus.
  • Anti-2019-nCoV N-IgY-pAbs showed neutralizing and inhibiting effect on 2019-nCoV.
  • the spray formulation formulated with anti-2019-nCoV N-IgY-pAbs can be stored stably at 4°C for at least 12 months.
  • the above application examples show that with the help of the above 20 amino acid sequences involved in this application, one or more of each amino acid sequence is used as the core, and then corresponding adjustments or modifications are made.
  • the modified materials include but are not limited to nanomaterials, fluorescent materials, Enzymes, biotin and specific proteins. It can be applied to the detection of novel coronavirus, including but not limited to ELISA detection, immunochemiluminescence detection and immunofluorescence detection. It can also be applied to the design of new coronavirus vaccine targets, including vaccine immune antigen design and vaccine performance evaluation.

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Abstract

具体公开了一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组及应用。靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组包括位于S-ECD区域的15种氨基酸序列和位于非结构蛋白(NSP)区域的5种氨基酸序列，可应用于新型冠状病毒的检测、治疗靶点设计和疫苗靶点设计。以上氨基酸序列组中，仅在1种S蛋白aa261-275序列中发现P272属于低频率突变残基，其余19种均属于保守氨基酸序列，不包含目前发现的病毒突变位点，具有高度保守性，可有效应对目前新冠病毒高频突变的不利形势。

Description

靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组及应用 技术领域
本申请涉及生物医药领域，更具体地说，它涉及一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs(neutralizing IgY polyclonal antibodies)的核心氨基酸序列组及应用。
背景技术
新型冠状病毒肺炎(简称新冠肺炎)疫情是当今全球最为关注的突发急性传染病事件，自疫情在全球爆发至今,还未得到有效地完全遏制。新型冠状病毒(简称新冠病毒)感染人数众多，传播性极强，病死率较高。截止目前为止，在新冠肺炎疫苗研制和治疗手段上有了长足进展，疫苗已经得到推广和上市应用，但仍尚无针对新冠病毒的特效治疗药物。因此，探索新型新冠肺炎疫情防御和诊治的新方法、新手段及新工具仍是迫切需要的。
基于病毒感染和产生抗体的基本免疫学机制，利用新型冠状病毒特异性抗体可以起到中和病毒作用，从而阻止病毒粘附、入侵宿主细胞。新冠病毒感染机体首先通过刺突蛋白(S蛋白)上的受体结合结构域(RBD)与宿主细胞的受体血管紧张素转换酶2(ACE2)结合，介导病毒进入宿主细胞。因此，在免疫原选择方面，采用新冠病毒S蛋白胞外区(S-ECD)是一个理想和有效的免疫抗原，可有效地诱导中和抗体的产生。
在抗体种类上，禽类或鸟类的IgY抗体具有优势。IgY被动免疫治疗策略曾用于人类和动物的病原体感染防御和治疗上。其中，病毒S蛋白是呼吸道冠状病毒抗体类药物优选的靶标蛋白。对于过去发生的呼吸道烈性病毒性传染病治疗药物开发中，例如中东呼吸综合征冠状病毒(MARS-CoV),有学者使用重组MERS-CoV S亚基蛋白制备了IgY多克隆抗体，并进行了体外中和试验及在体动物模型检测，均发现该IgY抗体能有效中和MERS-CoV的感染效果。还有研究采用核衣壳蛋白(N蛋白)制备的IgY多抗，也显示了强大的与N蛋白的高亲和能力。然而，IgY多抗识别新冠病毒蛋白组中哪些特异靶标位点仍然未知。
抗新冠病毒抗体通常可分为两大类，即中和抗体(NAbs，Neutralizing Antibodies)和非中和病毒结合抗体(BAbs,Binding Antibodies)。新冠病毒S蛋白是NAbs的关键靶点，因为新冠病毒是通过其S蛋白与宿主细胞表面ACE2蛋白的相互作用侵入宿主细胞的。而BAbs则可以与新冠病毒的所有蛋白质成分结合，包括S、N、E和M蛋白。NAbs是预测新冠疫苗成功与否的最重要标准之一。
在目前新冠病毒防治形势下，尽管新冠疫苗已经开发上市，用于应对新冠肺炎大流 行，但是，目前仍无特效的新冠治疗药物。在新冠疫苗和药物持续性开发过程中，目前面临的一个严重挑战是新冠病毒频繁出现突变，这些突变可导致病毒免疫逃逸或增强适应性。新突变株易引起疫情反复，削弱疫苗保护作用，从而恶化疫情全球蔓延形势。因此，针对病毒突变，在新冠病毒检测和治疗靶点设计上，应避开突变热点区域，选择保守区域和序列。
自2019年12月至2020年12月，新冠病毒以不同的速度发生着突变，研究学者分析了27个不同蛋白质在不同时期的突变率，发现新冠病毒不同蛋白的突变率是截然不同的，观察到新冠病毒疫苗和治疗中的S蛋白和N蛋白是具有最高的突变变异性，例如D614G(S蛋白)、P323L(NSP12)和R203K/G204R(N蛋白)。最近的突变又发现了其他位点，例如A222V(S蛋白)、L18F(S蛋白)和A220V(N蛋白)(Vilar S,Isom D G.One Year of SARS-CoV-2:How Much Has the Virus Changed？.Biology,2021,10:91)。对于近来的新冠病毒Delta变异株，其突变至少有13个位点，主要在S蛋白，例如D614G、T478K、L452R、P681R、E484Q(S蛋白)。
在新冠疫苗和治疗药物开发研究中，因为主要靶点和研究焦点在S蛋白和N蛋白上，而S蛋白和N蛋白具有最高的突变变异性，增加了在S蛋白和N蛋白上选择靶序列的难度，尤其是针对S蛋白。
因此，发明人认为，在抗体特异性结合靶序列评估中，是否存在突变区，是否包含突变点，是一项效能评价的重要指标，面对目前新冠病毒高频突变的不利形势，在S蛋白上发现与特异性抗体尤其是中和抗体结合的保守氨基酸序列有着重要的意义和应用价值。
发明内容
为了有效应对目前新冠病毒高频突变的不利形势，本申请提供一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组及应用。
第一方面，本申请提供的一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组采用如下的技术方案：
一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组，包括以下氨基酸序列：
位于S-ECD区域的氨基酸序列按照蛋白质序列编号如下：
S-NTD： 21RTQLPPAYTNSFTRG 35, 141LGVYYHKNNKSWMES 155, 261GAAAYYVGYLQPRTF 275和 291CALDPLSETKCTLKS 305；
S-RBD： 411APGQTGKIADYNYKL 425和 461LKPFERDISTEIYQA 475；
S-CTD1： 561PFQQFGRDIADTTDA 575, 571DTTDAVRDPQTLEIL 585和 581TLEILDITPCSFGGV 595；
S-CTD2： 661ECDIPIGAGICASYQ 675；
S1/S2交界区域： 741YICGDSTECSNLLLQ 755, 811KPSKRSFIEDLLFNK 825和 821LLFNKVTLADAGFIK 835；
S-HR2： 1161SPDVDLGDISGINAS 1175；
S-HR2-TM： 1201QELGKYEQYIKWPWY 1215；
位于非结构蛋白区域的氨基酸序列按照蛋白质序列编号如下：
ORF1ab： 1361SNEKQEILGTVSWNL 1375；
ORF1ab： 6411HHANEYRLYLDAYNM 6425；
ORF10： 21MNSRNYIAQVDVVNFNLT 38；
ORF7a： 1MKIILFLALITLATC 15和 111TLCFTLKRKTE 121。
在一些实施方案中，所述靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组由以下氨基酸序列组成：
位于S-ECD区域的氨基酸序列按照蛋白质序列编号如下：
S-NTD： 21RTQLPPAYTNSFTRG 35, 141LGVYYHKNNKSWMES 155, 261GAAAYYVGYLQPRTF 275和 291CALDPLSETKCTLKS 305；
S-RBD： 411APGQTGKIADYNYKL 425和 461LKPFERDISTEIYQA 475；
S-CTD1： 561PFQQFGRDIADTTDA 575, 571DTTDAVRDPQTLEIL 585和 581TLEILDITPCSFGGV 595；
S-CTD2： 661ECDIPIGAGICASYQ 675；
S1/S2交界区域： 741YICGDSTECSNLLLQ 755, 811KPSKRSFIEDLLFNK 825和 821LLFNKVTLADAGFIK 835；
S-HR2： 1161SPDVDLGDISGINAS 1175；
S-HR2-TM： 1201QELGKYEQYIKWPWY 1215；
位于非结构蛋白区域的氨基酸序列按照蛋白质序列编号如下：
ORF1ab： 1361SNEKQEILGTVSWNL 1375；
ORF1ab： 6411HHANEYRLYLDAYNM 6425；
ORF10： 21MNSRNYIAQVDVVNFNLT 38；
ORF7a： 1MKIILFLALITLATC 15和 111TLCFTLKRKTE 121。
通过三维蛋白质结构分析新冠病毒中的残基突变频率分级度(MRs)，将编码残基 从极低突变频率分级度到低突变频率分级度，分为3个等级，分别为MRs＝0.01–0.025，MRs＝0.025–0.05和MRs＝0.05–0.10；当MRs>0.20时，为高突变频率度。以上20种高效中和抗体识别的靶氨基酸序列，15种被鉴定位于S-ECD区域，5种位于非结构蛋白(NSP)区域。其中仅在1种S蛋白aa261-275中发现P272系属于低频率突变残基外，其余19种均属于保守氨基酸序列，不包含目前发现的病毒突变位点，具有高度保守性，可有效用于应对目前新冠病毒高频突变的不利形势。
可选的，氨基酸序列为进行相应调整或修饰后的氨基酸序列，修饰的材料包括纳米材料、荧光材料、酶类、生物素和蛋白质中的一种或几种。
通过采用上述技术方案，由于采用上述氨基酸序列中的一个或多个为核心，进行相应调整或修饰，修饰材料包括但不限于纳米材料、荧光材料、酶类、生物素及特异性蛋白质，有利于将调整或修饰的氨基酸序列应用于新冠病毒的检测、新冠病毒疫苗的免疫抗原设计和新冠病毒疫苗评价等。
第二方面，本申请提供一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组的应用，采用如下的技术方案：
一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组的应用，以各氨基酸序列中的一个或多个为核心，应用于新型冠状病毒的检测，或者应用于制备检测新型冠状病毒的试剂或试剂盒。
通过采用上述技术方案，上述氨基酸序列具有高特异性和高亲和力特性，通过各氨基酸序列中的一个或多个为核心，可以有效应用于新型冠状病毒的定量和/或定性检测。
可选的，所述检测包括但不限于ELISA检测、免疫化学发光法检测和免疫荧光法检测。
通过采用上述技术方案，各氨基酸序列可用于ELISA检测、免疫化学发光法检测和免疫荧光法检测等多种检测方法，应用范围广、适用性强。
一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组的应用，以各氨基酸序列中的一个或多个为核心，应用于新型冠状病毒治疗靶点设计。
通过采用上述技术方案，上述氨基酸序列具有高特异性和高亲和力特性，以各氨基酸序列中的一个或多个为核心，将其应用于新型冠状病毒治疗靶点设计，可以设计出针对新型冠状病毒的治疗制剂。
可选的，所述治疗靶点设计包括但不限于治疗性抗体的靶点设计和非抗体类治疗 性药物的靶点设计。
通过采用上述技术方案，各氨基酸序列可应用于治疗性抗体的靶点设计和非抗体类治疗性药物的靶点设计，应用范围广、适用性强。
一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组的应用，以各氨基酸序列中的一个或多个为核心，应用于新型冠状病毒疫苗靶点设计。
通过采用上述技术方案，上述氨基酸序列具有高特异性和高亲和力特性，以各氨基酸序列中的一个或多个为核心，可以应用到新型冠状病毒疫苗靶点设计中，有效用于应对目前新冠病毒高频突变的不利形势。
可选的，所述疫苗靶点设计包括但不限于疫苗免疫抗原设计和疫苗性能评价。
通过采用上述技术方案，各氨基酸序列可应用于疫苗免疫抗原设计和疫苗性能评价中，应用范围广、适用性强。
综上所述，本申请具有以下有益效果：
1、由于本申请提出的20种氨基酸序列中，仅有一种氨基酸序列中包含一个S蛋白极低频突变位点(S蛋白P272)，其余19种氨基酸序列均为保守序列，不包含目前发现的病毒突变位点，具有高度保守性，可有效用于应对目前新冠病毒高频突变的不利形势。
2、本申请中利用这20种高特异性和高亲和力的氨基酸序列，可有效设计新型冠状病毒检测试剂，以及针对新冠病毒的治疗制剂及疫苗制剂。
附图说明
图1是本申请实施例新冠病毒蛋白质组芯片检测图，检测抗新冠病毒中和抗体N-IgY-pAbs识别不同多肽结合位点的免疫应答反应，N-IgY-pAbs(375ng/ml)。
图2是本申请实施例新冠病毒蛋白质组芯片检测图，检测未免疫的母鸡血清识别不同多肽结合位点的免疫应答反应，未免疫的母鸡血清(1：2000)。
图3是本申请实施例新冠病毒蛋白质组芯片检测图，按照Z分值>0.05得到免疫应答反应分布图，Z分值>3.0被认为是显著的强信号，而Z分值≥5.0被视为显著的顶峰信号。
图4是S-ECD蛋白序列示意图(1273个氨基酸残基)。图中S-ECD显示：两个主要域S1(头部)和S2(茎区)。不同的结构域以不同颜色的标示，一级结构序列分布名称：SP(起始端)、NTD(N-末端结构域)；RBD(受体结合结构域)包含RBM(受体结合基序)、CTD1(C-末端结构域1)、CTD2(C-末端结构域2)、S1/S2边界(S1/S2交界处)、 S2'(S2'切割位点)、FL(融合环)、FPPR(融合肽近端区域)、HR1(七肽重复序列1)、CE(中心螺旋区)、CD(连接子结构域)、HR2(七肽重复序列2)、TM(跨膜结构域)和CT(C未端)。
图5是除S-ECD外，其他结构域中Z分值>0.05的氨基酸序列，其中非结构蛋白域中有5种氨基酸序列，Z分值>3.0被认为是显著的强信号。
具体实施方式
以下结合附图1-5和实施例对本申请作进一步详细说明。予以特殊说明的是：以下实施例中未注明具体条件者按照常规条件或制造商建议的条件进行，以下实施例中所用原料除特殊说明外均可来源于普通市售。
实施例
实施例1
1、抗新冠病毒中和抗体N-IgY-pAbs的制备：
采用公告号为CN112094341B的中国发明专利提出的抗新型冠状病毒的IgY中和抗体的制备方法，使用新型冠状病毒刺突蛋白胞外区(S-ECD蛋白)，免疫母鸡，提取卵黄抗体，筛选和制备抗新冠病毒中和抗体N-IgY-pAbs。
2、新型冠状病毒蛋白质组芯片的制备：
所有生物素标记肽由上海强耀生物科技有限公司和国平药业公司完成。所有新冠病毒E、N和S蛋白质均从北京义翘神州科技股份有限公司购置。多肽和蛋白质(表1)平行打印在3D改良玻片表面(由博奥生物提供)，并使用Arrayjet生物芯片点样仪进行点样，多肽芯片存储在-20℃，直到可以使用。
表1.载玻片矩阵上点样的对照品、多肽和蛋白质的序列区和名称
Figure PCTCN2022126859-appb-000001
Figure PCTCN2022126859-appb-000002
3、抗新型冠状病毒N-IgY-pAbs的结合靶点的全蛋白质组扫描：
(1)N-IgY-pAbs的杂交反应：将蛋白质组芯片置于培养盘中，并在室温下用含有5％(w/v)牛奶和0.2％(v/v)吐温-20的PBST封闭10分钟。洗涤后，将微阵列1和2分别与N-IgY-pAbs一起孵育(N-IgY-pAbs浓度分别为375ng/mL和186ng/mL)。同时，将免疫前母鸡血清添加到微阵列3(血清稀释2000x)和微阵列4(血清稀释4000x)，均孵育30分钟。随后，洗涤三次后，将芯片与与标记有Alexa Fluor 555的羊抗鸡二抗(美国Abcam)室温孵育下20分钟。然后，用真空泵干燥。
(2)数据分析：通过GenePix 4300A芯片扫描仪(美国Molecular Devices)扫描蛋白质组芯片，扫描及分析结果见图1-图5。
使用GenePix Pro7软件(美国Molecular Devices)提取每个斑点的荧光信号强度中位数。原始荧光信号强度为每个斑点的信号强度中位数减去每个斑点的背景强度中位数，然后计算重复孔的平均值。产生的信号用Z分值标准化。Z分值越高，表示反应信号越强，对抗新冠病毒中和抗体N-IgY-pAbs的识别越特异，亲和力越强。
Z分值>3.0被认为是显著的强信号，而Z分值≥5.0被视为显著的顶峰信号。结果表明，对照组中4个阴性对照是无信号，2个阳性对照中仅IgG和IgM的混合物显示强 阳性，但人脊髓灰质炎病毒多肽系无信号，说明抗新冠病毒中和抗体N-IgY-pAbs不识别人脊髓灰质炎病毒多肽，因人脊髓灰质炎病毒多肽与新型冠状病毒无关，所以无信号显示。抗新冠病毒中和抗体N-IgY-pAbs能显著识别S1+S2、S1和S2蛋白阳性质控品，达到显著顶峰信号(Z分值)≥5.0，验证了抗新冠病毒中和抗体N-IgY-pAbs的高灵敏度和高特异性。
参照图3，有20个强效靶点被确定为显著强信号(即Z分值≥3.0)，还有11个被确定为显著顶峰信号的高效靶标多肽(Z分值≥5.0)。
按Z分值大小进行排序，20种Z分值大于3.0的氨基酸序列在蛋白质组芯片上的反应信号见表2，其中，仅有一种氨基酸序列中包含一个S蛋白低频突变位点(MRs<0.025，S-NTD蛋白P272)，其余19种氨基酸序列均为高保守序列，不包含目前发现的病毒突变位点，可用于抗高突变频率的新冠病毒应对的设计、开发和研究。
表2.蛋白质组芯片上的反应信号及靶向识别N-IgY-pAbs的核心氨基酸序列信息
Figure PCTCN2022126859-appb-000003
Figure PCTCN2022126859-appb-000004
以上20种具有高效中和效果的氨基酸序列，15种被鉴定位于S-ECD区域，5种位于非结构蛋白(NSP)区域，各氨基酸序列详见表2。
应用例
应用例1
抗新冠病毒中和抗体N-IgY-pAbs对新冠病毒的抑制作用检测：
在生物安全3级(BSL-3)实验室条件下，使用新型冠状病毒活病毒进行中和活性检测：
1)将Vero细胞(10 5/mL)接种到96孔板，每孔100μl，CO 2培养箱中37℃过夜培养。当细胞密度达到80％-90％时，洗涤备用。
2)将纯化后不同浓度的抗新冠病毒中和抗体N-IgY-pAbs(747μg/mL；74.7μg/mL；7.47μg/mL；0.747μg/mL)分别与100TCID50的新冠活病毒混合均匀，制得抗体与病毒混合液，总体积分别为100μL，在37℃下孵育1小时。设DMEM培养基空白孔为阴性对照。
3)取96孔板备用Vero细胞，弃上清液，将第2)步100μL各抗体浓度的抗体与病毒混合液加入细胞中，然后添加100uL维持培养基(含2％胎牛血清的DMEM培养基)，37℃下培养48小时。
4)收集细胞培养上清液，根据试剂盒说明书(Roche高纯病毒RNA纯化试剂盒，商品编号：11859992001)提取总RNA，使用新型冠状病毒核酸检测试剂盒(荧光定量PCR法，中国上海伯杰医疗科技有限公司生产)检测总RNA中的新冠病毒拷贝数。新冠病毒 的引物和探针以开放阅读框1ab(ORF1ab)和核衣壳蛋白(N蛋白)为靶点，根据试剂盒厂商提供的公式计算样品中新冠病毒病毒灭活率见表3，N-IgY-pAbs优化后浓度为7μg/ml。
表3.中和试验新冠病毒病毒灭活率
Figure PCTCN2022126859-appb-000005
应用例2
抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂及其稳定性：
1)抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂的制备：制剂成分包含抗新冠病毒中和抗体N-IgY-pAbs；氯化钠或甘露醇；无菌去离子水或注射用水。其中抗新冠病毒中和抗体N-IgY-pAbs的含量为3-15μg/ml，添加氯化钠时，氯化钠质量浓度为0.9％；添加甘露醇时，甘露醇含量为10-30g/L。
本实施例中具体制备方法如下：
第一步：将抗新冠病毒中和抗体N-IgY-pAbs通过0.22μm的滤膜抽滤。
第二步：取10μg抗新冠病毒中和抗体N-IgY-pAbs溶液，加入无菌去离子水，使抗新冠病毒中和抗体N-IgY-pAbs溶液和无菌去离子水以1:3的比例稀释，加入氯化钠调至其终浓度为0.9％，调节pH值到7.4。
第三步：用0.22μm真空抽滤瓶抽滤，分装在无菌的分装瓶中。
2)稳定性评价：将抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂在4℃下储存6-12个月，本实施例中储存12个月。采用酶联免疫吸附试验(ELISA)评价其稳定性。具体步骤如下：
a)抗原包被：将抗原S蛋白RBD或S-ECD用包被液稀释至0.5ug/mL浓度，每孔100uL，4℃过夜包被；
b)弃液洗板后封闭：加入封闭液200uL/孔，37℃封闭1小时。
c)弃液洗板后抗体反应：样本用抗体稀释液进行梯度稀释，每孔100uL加入待检测抗体，37℃反应1小时；
d)洗板3次；
e)加入生物素化二抗：加入250倍稀释的生物素化二抗100uL/孔，37℃反应1小时；
f)洗板3次；
g)加入SA-HRP工作液100uL/孔，37℃反应1小时；
h)洗板3次；
i)显色：加入TMB显色液100uL/孔，37℃，10分钟；
j)加终止液70uL/孔终止反应；
k)酶标仪在450nm波长测定吸光度。
抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂稳定性数据如表4所示，结果表明，抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂在4℃下可稳定存放至少12个月，OD值未出现显著变化(p>0.05)。
表4.抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂稳定性
Figure PCTCN2022126859-appb-000006
应用例3
应用例2中制备的抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂对新冠病毒变异型Omicron病毒的中和效果。
经深圳市第三人民医院P3实验室鉴定，应用例2中制备的抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂对新冠Omicron病毒株病毒抑制率大于99％。鉴定方法包括如下步骤：通过焦点减少中和试验(FRNT)方法，检测该喷雾制剂对新冠病毒变异型Omicron 变种体外感染Vero E6细胞的中和作用。结果显示，在浓度为305μg/ml的情况下，平均中和活性为99.53％。
应用例4
应用例2中制备的抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂对新冠Delta病毒的中和效果。
经武汉大学病毒学国家重点实验室P3实验室鉴定，应用例2中制备的抗新冠病毒中和抗体N-IgY-pAbs喷雾制剂，釆用RT-PCR方法，通过N基因和ORF1ab基因定量测定N-IgY-pAbs对新冠Delta病毒株(B.1.617.2)病毒感染活性的抑制作用，结果显示，在浓度为305μg/ml情况下，病毒平均抑制率为99.94％。
全蛋白组芯片检测结果表明，该抗新型冠状病毒N-IgY-pAbs具有特异性识别多种靶氨基酸序列的特性，这些靶氨基酸序列能够相互协同，以促进抗新型冠状病毒N-IgY-pAbs与病毒受体结合域(RBD)相互作用，阻断RBD与宿主受体血管紧张素转换酶-2(ACE2)的结合,起到高效中和新型冠状病毒作用。
抗新型冠状病毒N-IgY-pAbs显示对新型冠状病毒具有中和抑制作用。采用抗新型冠状病毒N-IgY-pAbs配制的喷雾制剂可在4℃下稳定存放至少12个月。
由上述应用例表明，借助本申请涉及到的上述20种氨基酸序列，以各氨基酸序列中的一个或多个为核心，然后进行相应调整或修饰，修饰材料包括但不限于纳米材料、荧光材料、酶类、生物素和特异性蛋白质。可应用于新型冠状病毒的检测，检测包括但不限于ELISA检测、免疫化学发光法检测和免疫荧光法检测。还可应用于新型冠状病毒疫苗靶点设计，包括疫苗免疫抗原设计和疫苗性能评价。
借助本申请涉及到的上述20种氨基酸序列，以各氨基酸序列中的一个或多个为核心，可以应用于新型冠状病毒治疗靶点设计，包括治疗性抗体的靶点设计和非抗体类治疗性药物的靶点设计。
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。

Claims (10)

  1. 一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组，其特征在于，包括以下氨基酸序列：
    位于S-ECD区域的氨基酸序列按照蛋白质序列编号如下：
    S-NTD： 21RTQLPPAYTNSFTRG 35, 141LGVYYHKNNKSWMES 155, 261GAAAYYVGYLQPRTF 275和 291CALDPLSETKCTLKS 305；
    S-RBD： 411APGQTGKIADYNYKL 425和 461LKPFERDISTEIYQA 475；
    S-CTD1： 561PFQQFGRDIADTTDA 575, 571DTTDAVRDPQTLEIL 585和 581TLEILDITPCSFGGV 595；
    S-CTD2： 661ECDIPIGAGICASYQ 675；
    S1/S2交界区域： 741YICGDSTECSNLLLQ 755, 811KPSKRSFIEDLLFNK 825和 821LLFNKVTLADAGFIK 835；
    S-HR2： 1161SPDVDLGDISGINAS 1175；
    S-HR2-TM: 1201QELGKYEQYIKWPWY 1215；
    位于非结构蛋白区域的氨基酸序列按照蛋白质序列编号如下：
    ORF1ab： 1361SNEKQEILGTVSWNL 1375；
    ORF1ab： 6411HHANEYRLYLDAYNM 6425；
    ORF10： 21MNSRNYIAQVDVVNFNLT 38；
    ORF7a： 1MKIILFLALITLATC 15和 111TLCFTLKRKTE 121。
  2. 一种靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组，其特征在于所述核心氨基酸序列组由以下氨基酸序列组成：
    位于S-ECD区域的氨基酸序列按照蛋白质序列编号如下：
    S-NTD： 21RTQLPPAYTNSFTRG 35, 141LGVYYHKNNKSWMES 155, 261GAAAYYVGYLQPRTF 275和 291CALDPLSETKCTLKS 305；
    S-RBD： 411APGQTGKIADYNYKL 425和 461LKPFERDISTEIYQA 475；
    S-CTD1： 561PFQQFGRDIADTTDA 575, 571DTTDAVRDPQTLEIL 585和 581TLEILDITPCSFGGV 595；
    S-CTD2： 661ECDIPIGAGICASYQ 675；
    S1/S2交界区域： 741YICGDSTECSNLLLQ 755, 811KPSKRSFIEDLLFNK 825和 821LLFNKVTLADAGFIK 835；
    S-HR2： 1161SPDVDLGDISGINAS 1175；
    S-HR2-TM： 1201QELGKYEQYIKWPWY 1215；
    位于非结构蛋白区域的氨基酸序列按照蛋白质序列编号如下：
    ORF1ab： 1361SNEKQEILGTVSWNL 1375；
    ORF1ab： 6411HHANEYRLYLDAYNM 6425；
    ORF10： 21MNSRNYIAQVDVVNFNLT 38；
    ORF7a： 1MKIILFLALITLATC 15和 111TLCFTLKRKTE 121。
  3. 根据权利要求1或2所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组，其特征在于，氨基酸序列为调整或修饰后的氨基酸序列，修饰的材料包括纳米材料、荧光材料、酶、生物素和特异性蛋白质中的一种或几种。
  4. 根据权利要求1至3中任一项所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组用于检测新型冠状病毒的应用。
  5. 根据权利要求4所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组的应用，其特征在于，所述检测包括ELISA检测、免疫化学发光法检测和免疫荧光法检测。
  6. 根据权利要求1至3中任一项所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组用于制备检测新型冠状病毒的试剂或试剂盒的应用。
  7. 权利要求1所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组用于设计新型冠状病毒的治疗靶点的应用。
  8. 根据权利要求7所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组的应用，其特征在于，所述治疗靶点包括治疗性抗体的靶点和非抗体类治疗性药物的靶点。
  9. 权利要求1至3中任一项所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组用于设计新型冠状病毒的疫苗靶点的应用。
  10. 根据权利要求9所述的靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组的应用，其特征在于，所述疫苗靶点包括疫苗免疫抗原和疫苗性能评价。
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