WO2011137569A1 - 人源抗狂犬病毒糖蛋白中和性抗体rvfab3 - Google Patents

人源抗狂犬病毒糖蛋白中和性抗体rvfab3 Download PDF

Info

Publication number
WO2011137569A1
WO2011137569A1 PCT/CN2010/001574 CN2010001574W WO2011137569A1 WO 2011137569 A1 WO2011137569 A1 WO 2011137569A1 CN 2010001574 W CN2010001574 W CN 2010001574W WO 2011137569 A1 WO2011137569 A1 WO 2011137569A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
rabies virus
gene
human
fab
Prior art date
Application number
PCT/CN2010/001574
Other languages
English (en)
French (fr)
Inventor
梁米芳
孙丽娜
陈哲
李川
李德新
Original Assignee
中国疾病预防控制中心病毒病预防控制所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国疾病预防控制中心病毒病预防控制所 filed Critical 中国疾病预防控制中心病毒病预防控制所
Publication of WO2011137569A1 publication Critical patent/WO2011137569A1/zh

Links

Classifications

    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to genetically engineered antibody technology, in particular to a human anti-rabies virus glycoprotein neutralizing antibody; the present invention also relates to the preparation of the antibody for prevention or treatment Application in rabies drugs. Background technique
  • Rabies is a worldwide zoonosis caused by rabies virus, which kills 100% of the disease.
  • WHO World Health Organization
  • PRIG human rabies immune globulin
  • ERI Equine rabies immune globulin Due to the serious side effects of ERIG and the inhibition of antibody responses to certain vaccines, HRIG is expensive, limited in supply and potentially pathogenic. Therefore, it is our goal to prepare a passive immunological preparation with high efficiency, low cost and low side reaction.
  • Human or animal serum immunoglobulins containing specific antibodies have a long history of preventing and treating infectious diseases.
  • the in vitro antiviral neutralizing activity of monoclonal antibodies and the protection of the body against viral challenge in vivo have been proved by many experiments, such as murine anti-hepatitis A virus, hantavirus, measles virus, RSV virus, CMV virus and other neutralizing monoclonal antibodies can be 100% protect animals from viral attack.
  • the use of antigen-immunized animals to obtain polyclonal antiserum pathways has been a classic method of obtaining antibodies, but lacks specificity and homogeneity.
  • MCAbs monoclonal antibodies
  • VAG Vaccinia immune globulin
  • the rabies monoclonal antibody CR57 and CR4098 made a monoclonal antibody cocktail that neutralized 26 typical street strains. Protective experiments on animals have shown that the use of monoclonal antibody cocktails for treatment is feasible and superior (Goudsmit J, et al. 2006).
  • Antibody Drug As with the transformation of blood-borne vaccines to genetically engineered vaccines, there is an urgent need to replace genetically-derived VIG with genetically engineered antibodies, such as by chimeric antibody technology.
  • a first object of the present invention is to provide a human anti-rabies virus glycoprotein neutralizing antibody and an active fragment thereof.
  • a second object of the present invention is to provide a gene encoding the above antibody or an active fragment thereof.
  • a third object of the present invention is to provide the use of the above antibody and its active fragment for the preparation of a medicament for the prevention or treatment of rabies or a diagnostic reagent.
  • the invention utilizes the phage surface presentation technology to collect a plurality of peripheral blood lymphocytes of a vaccine injection with high titer rabies virus antibody, and constructs a human anti-rabies virus genetic engineering antibody library by genetic engineering means, and screens for specific anti-rabies disease.
  • the obtained Fab segment antibody was named RVFab3.
  • This recombinant antibody is determined by the hypervariable region (CDRs)-specific gene sequences present in the variable regions of the antibody light and heavy chain genes, and is specifically expressed in prokaryotic cells to specifically bind to the function of rabies virus.
  • CDRs hypervariable region
  • It specifically recognizes rabies virus particle antigens, and both target rabies virus glycoprotein G, and has obvious immunofluorescence reaction (IFA) and enzyme-linked immunosorbent assay (ELISA) reactions with rabies virus, and has a neutralizing activity against rabies virus infection.
  • the RVFab3-specific light chain and heavy chain variable region genes are derived from a specific enrichment screen for a human anti-rabies virus antibody gene pool, which is derived from the peripheral blood lymphocyte gene of the Chinese rabies virus vaccine.
  • the corresponding three CDR region sequence combinations of the light and heavy chain variable regions and the framework region sequences between the CDR regions constitute the sequence characteristics of each antibody variable region, and RVFab3 belongs to the antibody heavy chain family VH4.
  • the function of the antibody protein is determined by the specific nucleotide sequence and its complement in the CDR1, CDR2 and CDR3 of the determinant complementary region of the light chain and heavy chain variable regions of the antibody gene, and the corresponding amino acid sequences of the corresponding CDR regions constitute the antibody.
  • the specific antigen binding region determines the antigen binding characteristics of the antibody in the invention and the anti-rabies virus functional characteristics.
  • the amino acid detailed sequences of the antibody light chain and heavy chain variable regions which determine the function of neutralizing antibodies and their comparison results are shown in Table 1: Table 1
  • the amino acid sequence of the RVFab3 light chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 2.
  • the gene sequence encoding the light chain variable region of RVFab3 is shown in SEQ ID No. 3, and the gene sequence of the heavy chain variable region is shown in SEQ ID No. 4.
  • amino acid sequence for example, replaces an amino acid having similar properties in a non-hypervariable region, such as replacing Val at position 8 of the heavy chain VH sequence of RVFab3 with Ala.
  • the gene sequence encoding the above Fab fragment antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody.
  • Those skilled in the art can artificially engineer the gene according to the preference of the expression of the antibody host to improve the expression efficiency of the antibody.
  • the present invention recombines the light chain variable region and the heavy chain variable region of the above Fab antibody to obtain a single-chain antibody (ScFv) having a smaller molecular weight, which antibody can also specifically recognize a rabies virus surface antigen, and has intracellular The role of immunity.
  • Single-chain antibodies have strong penetrating power and are easy to enter local tissues.
  • the gene encoding the Fab antibody and the ScFv gene can be cloned into an expression vector, and then transformed into a host, and a Fab antibody and a single-chain antibody can be obtained by inducing expression.
  • the light chain coding gene and the heavy Fd fragment gene of the above Fab antibody can be cloned into a total anti-expression vector and introduced into a host cell to obtain a total anti-immunoglobulin expressing anti-rabies virus.
  • the light chain and heavy Fd fragment genes of the above Fab antibody RVFab3 are separately cloned into the whole antibody expression vector pAC-L-Fc and transfected into insect Sf9 cells, Secreted expression of whole antibodies was achieved using a baculovirus/insect cell system to obtain a full antibody RV IgG3.
  • the obtained antibodies were functionally identified by ELISA, IFA and SDS PAGE.
  • the results showed that the human IgG whole antibody RVIgG3 specifically binds to the aG strain and the CTN strain rabies virus particles, and expresses ERA with the baculovirus/insect cell system.
  • the CVS, CTN and aG strains have specific binding to the glycoproteins of the four rabies virus strains.
  • the full-antibody function was identified by the rabies virus rapid immunofluorescence inhibition assay (RFFIT).
  • RFFIT rabies virus rapid immunofluorescence inhibition assay
  • the present invention successfully obtains a human neutralizing antibody specific for a rabies virus glycoprotein by using a phage antibody library technology; using the human neutralizing anti-rabies virus glycoprotein genetically engineered antibody variable region gene obtained by the above, Fab
  • the antibody gene and the whole antibody gene under the characteristics of each of the above antibody genes can express and produce the antibody in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system or the modified gene containing the antibody gene based thereon Any other gene that obtains an antibody product that neutralizes rabies virus infection, and is made into a specific antibody drug for clinical use in the prevention and treatment of rabies.
  • Figure 1 is an immunofluorescence analysis of human rabies virus Fab antibody and ERA, CVS CTN and aG strain rabies virus glycoprotein;
  • Figure 2 is a SDS-PAGE electropherogram of purified IgG
  • Figure 3 shows an anti-rabies virus human IgG antibody against CVS-11 rabies virus rapid immunofluorescence inhibition assay.
  • Virus, cell, vector Rabies virus aG strain, CTN strain, CVS strain and ERA strain are provided by the Chinese Center for Disease Control and Prevention of the Chinese Center for Disease Control and Prevention.
  • the human immunization strain PM strain is an imported human vaccine strain, and the cell used for the in vitro neutralization experiment of rabies virus is BHK-21 (ATCC).
  • the strain was XLI-Blue (Stratagene, USA) and the vector was P Comb3 (40 kb) supplied by the Scripps Research Institute of the United States.
  • the baculovirus expression vector is pAC-L-Fc (PROGEN PR3003, Germany) (Liang, MF, Stefan, D., Li, DX, Queitsch, I., Li, W., and Bautz, EF Baculovirus expression cassette vectors for rapid production Of complete human IgG from phage displayselected antibody fragments. Journal of Immunological Methods. 247: 119-130. Insect cell Sf from the American Cell Culture Center
  • rabies virus glycoprotein PCR amplification of rabies virus aG strain, CTN strain, CVS strain and ERA strain provided by the Chinese Center for Disease Control and Prevention, respectively.
  • G glycoprotein
  • primers were designed to introduce a BamHI restriction site at both ends, and a 6-His tag was added to the 3' segment.
  • the purified amplified product was digested with BamHI, and the digested fragment was recovered and directly cloned into the baculovirus expression vector pAcUW51 which was also digested with BamHI, and the HA gene was identified by PCR under the control of the polyhedrin promoter.
  • the recombinant plasmid was obtained as: pAc-HA.
  • Recombinant baculovirus was prepared by transfecting the recombinant plasmid into insect cells for protein expression, and the expression was carried out using the BaculoGold co-transfection kit of Pharmogen, USA. The method of operation is as follows: After mixing 5Mg of recombinant plasmid DNA with 0.5 of BaculoGold linear DNA, the Sf cells with a growth density of 50% were transfected with the transfection reagent in the kit, and cultured at 27 ° C for 4 days, collected. The cells are titrated and amplified as a virus of the recombinant virus.
  • the rabies virus aG strain and the CTN strain were respectively infected with BHK-21 cells, and the supernatant was cultured. After formaldehyde inactivation and safety examination, the virus particles were purified by centrifugation for 3 h at 20 °C with a continuous sucrose density gradient of 35000 g (Beckman). SW28).
  • phage antibody library Lymphocytes were isolated from anticoagulant of vaccination with high titer rabies virus antibody using lymphocyte separation solution (Sigma, USA), and total cellular RNA was extracted with RNeasy Mini Kit (QIAGEN, Germany). The extracted RNA was reverse-transcribed into cDNA using the Invitrogen First-Strand Synthesis System for RT-PCR. Cat No. 18080-051 using Oligo-dT primers. Human antibody IgG1 heavy chain Fd and light chain Kappa and Lambda primers were used for PCR amplification of human light and heavy chain Fab genes. The PCR conditions were: 94 ° C lmin, 54 ° C 1 min, 72 ° C 2 min, 35 cycles.
  • the method of building the library is basically carried out according to the literature (Barbas, C. Fill., Kang, AS, and lamer, RA Assembly of combinatorial antibody libraries on phage surface: the genelll site. Proc.Natl.Acad.Sci.USA.1991;88( 18): 7978-7982). ,
  • Enrichment screening of phage antibody library and induction expression of Fab fragment antibody The screening antigen was an inactivated virus particle aG and CTN strain purified by ultracentrifugation. Dilute with 0.1m/L NaHC0 3 (pH8.6) solution, coat the immunotube, block with 4% skim milk-PBS, 37 V for 2 h, then add the above phage antibody library, 1 ml per tube, 37 ° C was incubated for 2 h, washed repeatedly with 5% Tween-20-TBS for 20 times, and finally neutralized with 1 ml of a pH 2. 2 glycine-hydrochloric acid eluate, and neutralized with a pH of 9.6 Tris.
  • the specific enrichment screening method and the induced expression of the Fab segment were basically carried out according to the literature (Barbas, C. Fill., Kang, AS, and larner, RA Assembly of combinatorial antibody libraries on phage). Surface: the genelll site. Proc. Natl. Acad. Sci. USA. 1991; 88(18): 7978-7982).
  • the antibody was coated with anti-human Fab antibody (Sigma, 1:2000 dilution) in a solution of 0.1 m/L NaHC0 3 (pH 9.6) on the plate, overnight at 4 ° C; 4% skim milk was blocked , 37 °C lh, added Fab antibody, 37 ° C lh; added enzyme-labeled anti-human Fab secondary antibody (US Sigma, 1: 2000 dilution), 37 ° C lh; color development liquid, 2M H 2 The reaction was terminated by S0 4 , and the absorbance A value was measured by a microplate reader.
  • IFA Indirect immunofluorescence assay: Sf cells were infected with recombinant baculovirus expressing rabies virus glycoprotein, and infected cells were harvested 4 to 5 days later to prepare recombinant rabies virus glycoprotein antigen tablets. The expressed Fab was added, incubated at 37 °C for 30 min, rinsed, added with FITC-labeled anti-Fab antibody (Sigma, USA), incubated at 37 °C for 30 min, washed, air-dried, and observed under a microscope.
  • nucleic acid sequence analysis of human Fab antibody variable region gene Plasmid DNA was prepared using Qiagen Miniprep Kit (QIAGEN, Germany) for nucleic acid sequence analysis.
  • the primers for the light and heavy chains are 5'-AAACTAGCTAGTCGCCAAGGA-3' (as shown in SEQ ID No. 5) and 5'-CCGCGGTGGCGGCCGCAAAT-3' (as shown in SEQ ID No. 6).
  • the sequencing results were compared with the IgG gene sequence in the Internet V-Base gene pool.
  • Transfection and recombinant virus infection and proliferation The BaculoGold co-transfection kit from Pharmogen, USA. The method of operation outlined as follows: The recombinant plasmid DNA BaculoGold Linear DNA 5 g and after mixing 0.5 ⁇ ⁇ , using transfection reagent kit transfection growth density of 50% of the Sf cells, 27 ° C after 4d culture The supernatant was collected and titrated and amplified as a virus of the recombinant virus. See the Baculovirus expression vector system manual (BD Biosciences Pharmingen, USA) for specific procedures.
  • RFFIT Human anti-rabies virus antibody rapid immunofluorescence inhibition assay
  • the fluorescent antibody can inhibit the highest dilution factor of ⁇ 50% of the antibody, which is the neutralizing antibody titer of the antibody to be tested. According to the Reed & Muench formula, the ED 50 of each antibody sample and the labeled product was calculated to obtain the titer of each antibody to be tested.
  • the light chain gene and the heavy chain Fd fragment were cloned into pAC-L-Fc according to the above methods 8 to 11 and transfected into insect Sf cells, and the secreted expression of the whole antibody was achieved by the baculovirus/insect cell system, and The mutant was subjected to immunological detection.
  • the phage antibody library was enriched and screened with purified rabies virus particle aG strain, and 6000 clones were randomly picked after 2 rounds of screening.
  • the anti-human Fab antibody (sigma company, 1:2 000 diluted use), rabies virus particle aG strain antigen coated 96-well plate, added to the test sample supernatant, with enzyme-labeled anti-human Fab secondary antibody (Sigma, 1: 2 000 dilutions used) detection.
  • the results showed that a total of 2984 human Fab-positive clones were obtained, as shown in Table 2.
  • 181 clones specifically bind to the rabies virus granule aG strain, of which 36 Fab clones were identified against rabies virus glycoproteins.
  • the phage antibody library was enriched and screened by the purified rabies virus particle CTN strain, and 2400 clones were randomly picked after 2 rounds of screening.
  • anti-human Fab antibodies Sigma, 1:2 000 diluted use
  • rabies virus particle aG strain antigen coated 96-well plate added to the supernatant of the test sample, and detected with enzyme-labeled anti-human Fab secondary antibody (Sigma, 1:2 000 diluted use).
  • enzyme-labeled anti-human Fab secondary antibody Sigma, 1:2 000 diluted use.
  • the results showed that a total of 1833 human Fab-positive clones were obtained, as shown in Table 3.
  • 1833 human-derived Fab-positive clones 79 clones specifically bind to the rabies virus particle CTN strain, and 34 of the Fab clones were identified as directed against rabies virus glycoproteins.
  • the DNASTAR sequence analysis software was used for analysis and comparison of the IgG sequences in the Internet V-Base gene pool.
  • 11 of them were different in sequence. Therefore, this study successfully screened and cloned 11 antibodies with different antibody light and heavy chain variable region sequences and their combinations.
  • the heavy chain variable regions were mainly classified into IgG VH4 and VH3 families, and their light chain variable regions were mainly classified.
  • 11 human Fab monoclonal antibodies that specifically bind to rabies virus glycoprotein were named RVFabl-l.
  • the sequences of RVFabl, RVab3, RVFab5, RVFab8 and RVFab9 were selected as follows: Table 4 Comparison of amino acid sequences of human anti-rabies virus glycoprotein Fab antibody variable region gene
  • VR variable region
  • VH heavy chain in VR (heavy chain variable region)
  • VL light chain in VR
  • the light chain and heavy chain Fd genes of five Fab antibodies (RVFabl, RVFab3, RVFab5> RVFab8, RVFab9) that have been functionally identified were cloned into the whole antibody expression vector pAC-L-Fc and transfected into insect Sf9 cells.
  • Secreted expression of whole antibodies was achieved using a baculovirus/insect cell system, designated RVIgGl, RVIgG3, RVIgG5, RVIgG8 and RVIgG9, respectively.
  • the expression supernatant was directly purified by Amersham's Protein-A affinity chromatography column.
  • the expression and purification of the whole antibody IgG were confirmed by SDS-PAGE. The results showed that the pure protein was obtained, and the antibody after melting was clearly observed.
  • heavy chain located at about 28kD, 55kD. as shown in picture 2.
  • the antibody level When the antibody level is equal to or higher than 0.5 IU/ml, the antibody has neutralizing activity, and thus the five human monoclonal antibodies obtained in the present study have neutralizing activity against rabies virus. Among them, the neutralizing activity of RVIgG9 was weak, and the other 4 strains had good neutralizing activity, and fully possessed the ability to neutralize the international standard attack strain CVS-11, as shown in Fig. 3.
  • the light chain gene and heavy chain gene modified based on RVIgG3 were cloned into pAC-L-Fc, and transfected into insect S cells, and full antibody secretion was achieved by baculovirus/insect cell system. Expression, the mutant RVIgG3 was obtained. Immunological detection of the mutant, indirect immunofluorescence experiments showed that RVFab3' can specifically target rabies virus glycoprotein G, and use rapid immunofluorescence inhibition assay to detect antibodies in vitro and international standard attack strain CVS-11 strain. The neutralization reaction showed that the properties were substantially the same as those of RVIgG3. Industrial applicability
  • the human neutralizing anti-rabies virus glycoprotein genetically engineered antibody variable region gene, the Fab antibody gene and the whole antibody gene under the characteristics of each of the above antibody genes obtained by the present invention can be used in prokaryotic cells, yeast cells, eukaryotic cells and The expression and production of this antibody in any recombinant system, or any other gene containing this antibody gene based on this, obtains an antibody product having a neutralizing rabies virus infection, and is clinically used for the prevention and treatment of rabies. Specific antibody drugs.

Description

人源抗狂犬病毒糖蛋白中和性抗体 RVFAB3 技术领域 本发明涉及基因工程抗体技术，特别是涉及一种人源抗狂犬病毒 糖蛋白中和性抗体；本发明还涉及该抗体在制备预防或治疗狂犬病的 药物中的应用。 背景技术
狂犬病是由狂犬病毒引起的世界性人兽共患病， 一旦发病 100 % 死亡。 目前世界上 87个国家有狂犬病报道， 每年约有 5万多人死于 狂犬病 （Knobel DL，et al. 2005 ) 。 狂犬病暴露后预防是防治狂犬病 的主要措施。 对于严重暴露的人， 世界卫生组织 (World Health Organization, WHO)建议釆用狂犬疫苗注射结合抗狂犬病毒免疫球蛋 白 （rabies immune globulin, RIG)的方法。 目前使用的两类 RIG为人 抗狂犬病毒免疫球蛋白 ( human rabies immune globulin, HRIG)和马 抗狂犬病毒免疫球蛋白 ( Equine rabies immune globulin, ERIG)。 由 于 ERIG 副反应比较严重， 而且对某些疫苗的抗体反应有抑制， 而 HRIG价格昂贵，供应量有限并且有潜在的病原威胁。 因此制备高效、 价廉、 副反应小的被动免疫制剂是我们的目标。
含有特异性抗体的人源或动物血清免疫球蛋白用以预防和治疗 传染病已历史悠久。单克隆抗体的体外抗病毒中和活性和体内保护肌 体抵抗病毒攻击已获得许多实验证明， 如鼠抗甲肝病毒、 汉坦病毒、 麻疹病毒、 RSV病毒、 CMV病毒等中和性单克隆抗体可以在体内 100 %保护动物免受病毒攻击。以抗原免疫动物获得多抗血清的途径一直 是获得抗体的经典方法，但缺乏特异性和均一性。继而建立的 B淋巴 细胞杂交瘤技术使得众多科学家通过细胞工程可以在体外定向地制 备各种单克隆抗体（monoclonal antibody, McAb ) , 其特异性强， 性 质均一， 易于大量生产。 然而 McAb 多为鼠源性， 鼠源 McAb 的异 源性反应极大地限制了 McAb作为治疗制剂在人体的应用。 免疫球 蛋白（ Vaccinia immune globulin, VIG )作为抗体成分主要来自捐献者
(恢复期病人）免疫血清， 从获得阳性血清到通过安全性检测均需花 费大量的人力和财力， 这就使其大量制备受到限制， 同时由于来源于 血清所以容易发生血源性传播疾病的感染。因此使用人源基因工程产 品替代血制品则可克服这些缺陷，而人源基因工程抗体研究的不断深 入， 给这一领域的生物制品发展带来了新的希望和广阔前景。 通过抗 体分子基因水平的重组可获得多种多样的特异性鼠源及人源抗体，使 对单克隆抗体的研究有了突破性进展并越来越显示出其重要意义及 实际运用前景。人源抗狂犬病毒单克隆抗体的研制和噬菌体抗体库技 术的产生为解决被动免疫制剂问题提供了新的思路。狂犬单克隆抗体 CR57和 CR4098做成的单克隆抗体鸡尾酒中和了 26种典型的街毒 株。 对动物的保护性实验表明， 利用单克隆抗体鸡尾酒进行治疗具有 可行性和优越性（ Goudsmit J，et al. 2006 )。
80年代末 90年代初兴起的噬菌体抗体基因库技术兴起和整个基 因工程抗体技术研究领域的发展，使当今世界人源或基因工程抗体的 开发研究取得很大进展并已由基础研究阶段步入实质性应用研究和 开发阶段。人源抗病毒基因工程抗体，尤其是人源全抗体的研究成功， 给各种病毒性传染病的特异性预防和治疗带来了新的希望，在抗病毒 感染生物药领域逐渐形成了一类新的抗病毒药， 即所谓的抗体药
( Antibody Drug )。 如同当初血源性疫苗向基因工程疫苗的转变， 现 在也急需用基因工程抗体替代血源性 VIG， 如通过嵌合抗体技术
( Boulianne,G.L. et al.,1984; Morrison,S.L. et al.,1984 ) 、 人源化抗体 技术 （Jones，P.T. et al.,1986) , 携带人单抗的转基因小鼠技术 (Green,L.L. et al.,1994) 异体杂交瘤技术 （James，K.et al.，1987)、 噬菌 体表面展示技术 （Barbas，C.F. et al.,1991) 等产生人源化抗体， 现已成 为国内外研究的重大方向， 并正在逐步走向成功。 发明内容
本发明的第一个目的在于提供一种人源抗狂犬病毒糖蛋白中和 性抗体及其活性片段。
本发明的第二个目的在于提供编码上述抗体或其活性片段的基 因。
本发明的第三个目的在于提供上述抗体及其活性片段在制备预 防或治疗狂犬病药物或诊断试剂中的应用。
本发明运用噬菌体表面呈现技术，釆集多个具有高滴度狂犬病毒 抗体的疫苗注射者外周血淋 细胞，通过基因工程手段构建了人源抗 狂犬病毒基因工程抗体文库，并筛选获得特异抗狂犬病毒基因工程抗 体 Fab段。 获得的 Fab段抗体命名为 RVFab3。
这株重组抗体是由存在于抗体轻链和重链基因可变区中的高变 区 （CDRs ) 特异性基因序列决定的， 并在原核细胞中获得有效表达 的特异性结合狂犬病毒的功能性抗体。它特异性识别狂犬病毒颗粒抗 原， 且均针对狂犬病毒糖蛋白 G, 与狂犬病毒具有明显的免疫荧光反 应 （IFA )和酶联免疫（ELISA )反应， 具有抗狂犬病毒感染的中和 活性功能。
RVFab3特异性的轻链和重链可变区基因来源于对人源抗狂犬病 毒抗体基因库的特异性富积筛选，该抗体库的建立来源于中国狂犬病 毒疫苗免疫者外周血淋巴细胞基因。其轻链和重链可变区相应的三个 CDR区序列组合及其 CDR区之间框架区序列组成了每个抗体可变区 序列特征， RVFab3隶属于抗体重链家族 VH4。 抗体蛋白功能由存在 于抗体基因轻链和重链可变区的决定族互补区域 CDR1、 CDR2 和 CDR3中特异性核苷酸序列及其互补所决定， 6个相应的 CDR区氨基 酸序列构成了抗体的特异性抗原结合区域，决定发明中抗体的抗原结 合特征和抗狂犬病毒功能特征。决定中和抗体功能的抗体轻链和重链 可变区氨基酸详细序列及其比较结果如表 1所示： 表 1
Figure imgf000005_0001
RVFab3轻链可变区的氨基酸序列如 SEQ ID No.l所示， 其重链 可变区的氨基酸序列如 SEQ ID No.2所示。
编码 RVFab3轻链可变区的基因序列如 SEQ ID No.3所示， 重链 可变区的基因序列如 SEQ ID No.4所示。
应当理解， 在不影响 Fab抗体活性的前提下， 本领域技术人员可 对 SEQ ID No.1-2所示的氨基酸序列进行各种取代、添加和 /或缺失一 个或几个氨基酸获得具有同等功能的氨基酸序列，例如在非高变区将 具有类似性质的氨基酸进行替换， 如将 RVFab3的重链 VH序列的第 8位的 Val替换为 Ala。
此外， 考虑到密码子的简并性， 例如可在其编码区， 在不改变氨 基酸序列的条件下， 对编码上述 Fab段抗体的基因序列进行修改， 获 得编码相同抗体的基因。本领域技术人员可以根据表达抗体宿主的密 码子偏爱性， 人工合成改造基因， 以提高抗体的表达效率。
进一步，本发明将上述 Fab抗体的轻链可变区和重链可变区进行 重组， 获得分子量更小的单链抗体（ScFv )， 该抗体同样能够特异性 识别狂犬病毒表面抗原，具有细胞内免疫的作用。单链抗体穿透力强， 易于进入局部组织发挥作用。
可将上述编码 Fab抗体的基因、 ScFv基因克隆到表达载体中， 进而转化宿主， 通过诱导表达获得 Fab抗体以及单链抗体。
此外， 可将上述 Fab抗体的轻链编码基因和重 Fd段基因克隆到 全抗表达载体中， 并导入宿主细胞中， 获得表达抗狂犬病毒的全抗免 疫球蛋白。
在本发明的实施例中， 将上述 Fab抗体 RVFab3的轻链和重 Fd 段基因分别克隆入全抗体表达载体 pAC-L-Fc并转染昆虫 Sf9细胞， 利用杆状病毒 /昆虫细胞系统实现了全抗体的分泌型表达， 得到全抗 体 RVIgG3。
利用 ELISA、 IFA、 SDS PAGE对获得的全抗体进行功能鉴定, 结果表明人源 IgG全抗体 RVIgG3针对 aG株和 CTN株狂犬病毒颗 粒均有特异性结合，与杆状病毒 /昆虫细胞系统表达 ERA、 CVS、 CTN 和 aG株四种狂犬病毒株糖蛋白均有特异性结合。 利用狂犬病毒快速 免疫荧光灶抑制实验（RFFIT )对全抗体进行功能鉴定， 结果表明： RVIgG3具有较好的中和活性， 能达到 813.3IU/mg, 完全具备了中和 国际标准攻击毒株 CVS-11株的能力。
本发明运用噬菌体抗体库技术，成功地获得了特异性针对狂犬病 毒糖蛋白的人源中和性抗体；利用上述获得的人源中和性抗狂犬病毒 糖蛋白基因工程抗体可变区基因、 Fab抗体基因以及上述每个抗体基 因特征下的全抗体基因， 可以在原核细胞、 酵母细胞、 真核细胞及任 何重组系统中表达和生产此抗体或以此为基础的改建后的含有此抗 体基因的任何其他基因， 获得具有中和狂犬病毒感染的抗体产物， 制 成临床上用于预防和治疗狂犬病的特异性抗体药物。 附图说明
. 图 1是抗狂犬病毒人源 Fab抗体与 ERA、 CVS CTN和 aG株狂 犬病毒糖蛋白免疫荧光分析；
图 2是纯化后 IgG 的 SDS-PAGE电泳图；
图 3是抗狂犬病毒人源 IgG抗体针对 CVS-11狂犬病毒快速免疫 荧光灶抑制实验。 具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细 描述。 以下实施例用于说明本发明， 但不用来限制本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟 知的常规手段。 材料和方法
1.病毒、 细胞、 载体： 狂犬病毒 aG株， CTN株， CVS株和 ERA 株由中国疾病预防控制中心病毒病预防控制所提供。 人免疫用毒株 PM 株为进口人用疫苗株， 用于狂犬病毒体外中和实验的细胞为 BHK-21 ( ATCC ) 。 菌株为 XLI-Blue (美国 Stratagene ) ， 载体为 PComb3(40kb), 由美国 Scripps研究所提供。 杆状病毒表达载体为 pAC-L-Fc (德国 PROGEN PR3003 ) ( Liang, M. F., Stefan, D., Li, D. X.， Queitsch, I., Li, W., and Bautz, E. F. Baculovirusexpression cassette vectors for rapid production of complete human IgG from phage displayselected antibody fragments. Journal of Immunological Methods.247: 119-130. ) 。 昆虫细胞 Sf 来自美国细胞培养中心
( ATCC ) 。
2.抗原制备
2.1真核表达狂犬病毒糖蛋白 （ERA、 CVS, CTN, aG株） 分别利用中国疾病预防控制中心病毒病所提供的狂犬病毒 aG株， CTN株， CVS株和 ERA株的 cDNA为模板进行 PCR扩增糖蛋白 (G) 基因， 设计引物时 2端均引入 BamHI酶切位点， 并且在 3 ' 段添加 6-His标签。 纯化的扩增产物用 BamHI酶切， 将酶切后的片段回收， 直接克隆到同样经 BamHI酶切的杆状病毒表达载体 pAcUW51 中， 使 HA基因在多角体启动子的控制下， 经 PCR鉴定目的基因插入方 向后， 获得重组质粒为： pAc-HA。 通过将重组质粒转染昆虫细胞制 备重组杆状病毒进行蛋白表达， 表达釆用美国 Pharmogen 公司的 BaculoGold共转染试剂盒。 操作方法略述如下： 将 5Mg的重组质粒 DNA与 0.5 的 BaculoGold 线性 DNA 混合混合后，利用试剂盒中 的转染试剂转染生长密度为 50 %的 Sf 细胞， 27°C培养 4天后， 收集 上清作为重组病毒的毒种进行滴定和扩增。 具体操作见 Baculovirus expression vector system 手册。 获得的表达狂犬病毒糖蛋白 （ ERA、 CVS, CTN、 aG株）重组杆状病毒感染 Sf 细胞， 4~5天后收获感染 细胞制成重组狂犬病毒糖蛋白 （ERA、 CVS、 CTN、 aG株）抗原片。
2.2狂犬病毒纯化
分别收获狂犬病毒 aG株和 CTN株感染 BHK-21细胞后培养上 清， 经甲醛灭活和安全检查后，用 20%~50%连续蔗糖密度梯度 35000g, 4°C离心 3h纯化病毒颗粒 （Beckman SW28)。
3.噬菌体抗体库的构建： 用淋巴细胞分离液 （美国 Sigma )从具 有高滴度狂犬病毒抗体的疫苗注射者的抗凝血液中分离淋巴细胞，用 RNeasy Mini Kit (德国 QIAGEN)提取总细胞 RNA， 用 Oligo-dT引 物将提取的 RNA 釆用 Invitrogen 公司的第一链合成试剂盒 (SuperScriptTMlII First-Strand Synthesis System for RT-PCR. Cat No.18080-051)逆转录成 cDNA， 用一组扩增人源抗体 IgGl 重链 Fd 及轻链 Kappa和 Lambda 引物， 对人源轻链和重链 Fab基因进行 PCR扩增。 PCR条件为：94°C lmin, 54°C 1 min, 72°C 2 min, 35个 循环。 建库方法基本按文献进行（ Barbas, C. Fill., Kang, A. S.， and lamer, R. A. Assembly of combinatorial antibody libraries on phage surface: the genelll site. Proc.Natl.Acad.Sci.USA.1991;88(18): 7978-7982 ) 。 、
4.噬菌体抗体库的富集筛选及 Fab段抗体的诱导表达： 筛选抗原 为超速离心纯化的灭活病毒颗粒 aG和 CTN株。 使用时用 0.1m/L NaHC03 (pH8.6) 的溶液稀释，包被免疫管，用 4 %脱脂奶- PBS, 37 V 封闭 2 h后， 加入上述噬菌体抗体库， 每管 1ml, 37 °C孵育 2 h， 用 5 % Tween-20-TBS 反复洗 20 遍， 最后用每管 1ml pH2. 2 的甘氨酸- 盐酸洗脱液洗脱， pH9. 6 的 Tris 液中和。 洗脱后的噬菌体继续感染 2ml 新鲜的 OD6。。= 1. 0左右的 XLl-Blu菌， 经辅助噬菌体 VCSM13 (美国 Stratagene) 感染后进行下一轮筛选。 如此反复筛选 4~5 次。 具 体富集筛选方法及 Fab段的诱导表达基本按文献进行（Barbas, C. Fill., Kang, A. S.， and larner, R. A. Assembly of combinatorial antibody libraries on phage surface: the genelll site. Proc.Natl.Acad.Sci.USA.1991;88(18): 7978-7982 ) 。
5.人源抗狂犬病毒 Fab抗体的 ELISA检测
5.1 Fab表达的检测 用 0.1 m/L NaHC03 ( pH9.6 ) 的溶液包被 抗人 Fab抗体（美国 Sigma, 1： 2000稀释使用）于酶标板， 4°C过夜； 4%脱脂奶封闭， 37°C lh, 加入表达的 Fab抗体， 37°C lh; 加入酶标 抗人 Fab二抗（美国 Sigma, 1 ： 2000稀释使用）， 37°C lh; 显色液显 色， 2M H2S04终止反应， 酶标仪检测吸光度 A值。
5.2 间接酶联免疫法检测 Fab 与狂犬病毒结合活性 用纯化的 灭活狂犬病毒颗粒作为包被抗原， 随后的步骤同上。
6.间接免疫荧光 （IFA )检测： 利用已构建的表达狂犬病毒糖蛋 白的重组杆状病毒感染 Sf 细胞， 4~5d后收获感染细胞制成重组狂 犬病毒糖蛋白抗原片。 加入表达的 Fab, 37°C温育 30 min， 冲洗， 加 入 FITC标记的抗 Fab 抗体（美国 Sigma ) ， 37°C温育 30 min, 冲 洗， 晾干， 显微镜下观察。
7.人源 Fab抗体可变区基因的核酸序列分析： 用 Qiagen Miniprep Kit (德国 QIAGEN) 制备质粒 DNA进行核酸序列分析。 轻重链的测 序引物分别为 5' - AAACTAGCTAGTCGCCAAGGA-3 ' (如 SEQ ID No.5所示）和 5' -CCGCGGTGGCGGCCGCAAAT -3' (如 SEQ ID No.6 所示） 。 测序结果和 Internet V-Base基因库中 IgG基因序列比较。
8.全抗体重组表达质粒的构建： 将获得的 Fab 抗体的轻链先用 Xba l单酶切， 37°C3h, 然后用 Klenow酶进行补平， 37°C2h, 回收目 的片段； 再用 Sac I对目的片段进行酶切， 37°C3h, 此时琼脂糖凝胶 电泳中所见的大约 700bp的片段即轻链片段， 回收轻链片段， 克隆入 经 SacI/EcoRV 37°C3h双酶切后的 pAC-L-Fc 载体（德国 PROGEN PR3003 ) ( Liang, M. F" Stefan, D.， Li, D. X., Queitsch, I., Li, W., and Bautz, E. F. Baculovirusexpression cassette vectors for rapid production of complete human IgG from phage displayselected antibody fragments. Journal of Immunological Methods.247: 119-130. ) ,再将重链 Fd段利用 Xhol/Spel位点克隆进去， 构建成全抗体表达载体。
9.转染及重组病毒感染与增殖： 釆用美国 Pharmogen 公司的 BaculoGold共转染试剂盒。 操作方法略述如下： 将 5 g的重组质粒 DNA与 0.5μβ的 BaculoGold 线性 DNA .混合后， 利用试剂盒中的 转染试剂转染生长密度为 50 %的 Sf 细胞， 27°C培养 4d后， 收集上 清作为重组病毒的毒种进行滴定和扩增。 具体操作见 Baculovirus expression vector system 手册 (BD Biosciences Pharmingen, USA)。
10.全抗体 IgG分泌表达和纯化： 重组病毒感染生长密度 70 %左 右的 Sf 细胞， 27°C吸附 lh. 改用 SF-900 II SFM 无血清培养液， 27°C培养 3 ~ 5d后收集上清。 釆用 Amersham公司的 Protein- A亲和层 析柱直接纯化表达上清 ( Harlow E, Lane D. Antibodies: A Laboratory Manual [M]. New York: Cold Spring Harbor Laboratory Press , 1988. ) 。 利用 ELISA及 IFA对所获 纯化的 IgG抗体功能特性进行鉴定。具体操作见材料方法 5、 6部分。
11.人源抗狂犬病毒抗体快速免疫荧光灶抑制实验（RFFIT )： 取. 抗体及抗体标准品各稀释度 50μ1 于 96孔培养板中，加入中和用病毒 CVS-11 , 50μ1/孔， 同时设空白孔对照， 以及中和用病毒对照孔， 混 匀后置 37°C中和 1小时，每孔加入 lxl06 /ml BHK-21细胞悬液 50μ1， 置 37°C 5% C02孵箱中培养 24小时。 待培养结束吸干培养液， 每孔 中加入 ΙΟΟμΙ/ PBS清洗并吸干后， 每孔加入预冷至 4°C的 80 %丙酮 50 μΐ, -30°C固定 10分钟， 弃丙酮， 待挥发干燥后加入工作浓度的荧 光标记抗狂犬病病毒核蛋白抗体， 50μ1 /孔， 37°C孵育 30分钟， 甩掉 液体， 用 PBS洗板 2 ~ 3次， 甩干液体， 每孔加入 80 %甘油 50μ1, 荧 光显微镜观察。实验组中能使荧光灶抑制≥50 %的抗体最高稀释倍数, 即为被检抗体的中和抗体滴度。根据 Reed & Muench公式， 计算各 抗体样品及标注品的 ED50， 从而得出各待检抗体的效价。
12.非高变区突变后的抗体对狂犬病毒抗性的研究 基于 RVIgG3重链可变区氨基酸序列， 将 SEQ ID No.2所示氨基 酸序列的第 8位的 Val替换为 Ala, 将 RVIgG3轻链可变区（ SEQ ID No.l所示） 的第 10位的 Gly替换为 Gln。 分别合成 RVIgG3的重链 编码核酸序列 （在相应位置将密码子 gtg替换为 gcc ) 以及轻链编码 核酸序列 （在相应位置将密码子 ggg替换为 cag ) 。 按照上述 8~11 的方法，将轻链基因和重链 Fd段克隆到 pAC-L-Fc中，并转染昆虫 Sf 细胞， 利用杆状病毒 /昆虫细胞系统实现全抗体的分泌型表达， 并对 该突变体进行免疫学检测。 结果
1.人源抗狂犬病毒抗体库的筛选
用纯化的狂犬病毒颗粒 aG株对噬菌体抗体库进行富集筛选， 2 轮筛选后随机挑取 6000个克隆。用抗人 Fab抗体（ sigma公司， 1:2 000 稀释使用）、 狂犬病毒颗粒 aG株抗原包被 96孔板， 加入待测样品上 清， 用酶标抗人 Fab 二抗（Sigma公司， 1:2 000稀释使用）检测。 结果显示共获得 2984株人源 Fab表达阳性克隆， 如表 2所示。 2984 个人源 Fab表达阳性克隆中， 有 181个克隆对狂犬病毒颗粒 aG株特 异性结合， 其中 36株 Fab克隆被确定为针对狂犬病毒糖蛋白。
Figure imgf000011_0001
用纯化的狂犬病毒颗粒 CTN株对噬菌体抗体库进行富集筛选， 经 2轮筛选后随机挑取 2400个克隆。 用抗人 Fab抗体（sigma公司, 1:2 000稀释使用） 、 狂犬病毒颗粒 aG株抗原包被 96孔板， 加入待 测样品上清， 用酶标抗人 Fab二抗（Sigma公司， 1:2 000稀释使用） 检测。结果显示共获得 1833株人源 Fab表达阳性克隆，如表 3所示。 1833个人源 Fab表达阳性克隆中，有 79个克隆对狂犬病毒颗粒 CTN 株特异性结合， 其中 34株 Fab克隆被确定为针对狂犬病毒糖蛋白。
CTN株对噬菌体抗体库富集筛选
Figure imgf000012_0001
2.人源抗狂犬病毒 Fab抗体的序列分析
用 DNASTAR 序列分析软件进行分析处理， 比较 Internet V-Base基因库中的 IgG序列， 上述 70株特异性结合狂犬病毒糖蛋 白的人源 Fab单克隆抗体中， 其中有 11株序列不同。 因此本研究成 功筛选并克隆 11株带有不同的抗体轻重链可变区序列及其组合的抗 体， 其重链可变区主要分类在 IgG VH4 和 VH3 家族， 其轻链可变 区主要分类在 IgG VL1、 VL2、 VL3和 VK1 家族， 将 11株特异性 结合狂犬病毒糖蛋白的人源 Fab单克隆抗体命名为 RVFabl-l l。其中 选取 RVFabl、 RVab3、 RVFab5、 RVFab8和 RVFab9比较序列如下： 表 4人源抗狂犬病毒糖蛋白 Fab抗体可变区基因的氨基酸序列比较
Figure imgf000012_0002
RVFab5 VH SVNFYWS YIYYSGSTNYNPSLKS ERLTTGAMDV
VL SGSSSNIGSNYVS DN KRPS GTWDSSLSAWV
RVFab8 VH SVNSYWG NFYYSGNTHYNPSL S QSTIGGFFDY
VL TGTSSDIGNYNLVS EVT RPS SSYTATKNYWI VFab9 VH SVNSYWG NFYYSGNTHYNPSLKS QSTIGGFFDY
VL TGTSSDVGSYNLVS EGSKRPS CSYAGSSTWV
VR, variable region (可变区）； VH, heavy chain in VR (重链可变区）； VL, light chain in VR
(轻链可变区） . '
CDR, complementarity determining region (互补决定区） .
3.人源抗狂犬病毒 Fab抗体对狂犬病毒糖蛋白的特异性结合 为了证实所获得的重组 Fab抗体对不同狂犬病毒株糖蛋白的结 合特异性， 我们进一步通过间接免疫荧光试验（IFA )鉴定原核表达 Fab抗体的功能活性。 如图 1所示， RVFabl与 ERA、 CVS CTN和 aG株四种狂犬病毒毒株糖蛋白免疫荧光均为阳性，与正常 sf9细胞对 照反应为阴性，其余 10株人源 Fab抗体与不同狂犬毒株糖蛋白免疫 荧光结果与 RVFabl 相同。 免疫荧光结果证明从抗体库筛选获得的 11株 Fab阳性抗体均针对狂犬病毒糖蛋白， 且反应谱较广。
4.全抗体 IgG表达和纯化
将 5株已完成功能鉴定的 Fab抗体（ RVFabl、 RVFab3、 RVFab5> RVFab8、 RVFab9 ) 的轻链和重链 Fd段基因， 分别克隆入全抗体表 达载体 pAC-L-Fc后转染昆虫 Sf9细胞， 利用杆状病毒 /昆虫细胞系统 实现全抗体的分泌型表达，分别命名为 RVIgGl , RVIgG3, RVIgG5, RVIgG8及 RVIgG9。 釆用 Amersham公司的 Protein-A 亲和层析柱 直接纯化表达上清， 通过 SDS-PAGE检验全抗体 IgG的表达及纯化 情况，结果证实得到较纯蛋白，可清晰观察到解链后的抗体轻、重链， 分别位于约 28kD、 55kD处。 如图 2所示。
5.人源抗狂犬病毒抗体快速免疫荧光灶抑制实验（RFFIT ) 为了进一步研究 5株全抗体 IgG ( RV G1、 RVIgG3、 RVIgG5、 RVIgG8、 RVIgG9 )的中和活性， 我们釆用了快速免疫荧光灶抑制实 验来检测抗体在体外与国际标准攻击毒株 CVS-11株的中和反应， 结 果显示 5株人源抗狂犬病毒全抗体 RVIgG 1、 RVIgG3、 RVIgG5、 RVIgG 及 RVIgG9的中和效价分别为 866.6 IU/mg, 813.3 IU/mg, 689.8 IU/mg, 876.6 IU/mg ,428.5 IU/mg, 根据 WHO狂犬病专家委员会评定中和抗 体水平等于或高于 0.5IU/ml 即表示该抗体具有中和活性， 因此本研 究所获得的 5 株人源单抗针对狂犬病毒均具有中和活性。 其中 RVIgG9的中和活性较弱， 其余 4株具有较好的中和活性， 完全具备 了中和国际标准攻击毒株 CVS-11株的能力， 如图 3所示。
6.非高变区突变后的抗体对对狂犬病毒抗性影响
按照上述 8~11的方法， 将基于 RVIgG3修改后的轻链基因和重 链基因克隆到 pAC-L-Fc 中， 并转染昆虫 S 细胞， 利用杆状病毒 / 昆虫细胞系统实现全抗体的分泌型表达， 得到突变体 RVIgG3，。 对该 突变体进行免疫学检测， 间接免疫荧光实验表明 RVFab3'能特异性针 对狂犬病毒糖蛋白 G， 釆用了快速免疫荧光灶抑制实验来检测抗体 在体外与国际标准攻击毒株 CVS-11株的中和反应， 结果显示其性质 与 RVIgG3基本相同。 工业实用性
本发明获得的人源中和性抗狂犬病毒糖蛋白基因工程抗体可变 区基因、 Fab抗体基因以及上述每个抗体基因特征下的全抗体基因， 可以在原核细胞、 酵母细胞、 真核细胞及任何重组系统中表达和生产 此抗体， 或以此为基础的改建后的含有此抗体基因的任何其他基因， 获得具有中和狂犬病毒感染的抗体产物，制成临床上用于预防和治疗 狂犬病的特异性抗体药物。

Claims

权 利 要 求 书
1、 一种人源抗狂犬病毒糖蛋白中和性抗体， 其特征在于， 其轻. 链 CDR1、 CDR2和 CDR3以及重链 CDR1、 CDR2和 CDR3的氨基 酸序列如下表所示：
Figure imgf000015_0001
2、 如权利要求 1所述的抗体， 其特征在于， 其轻链可变区氨基 酸序列和重链可变区氨基酸序列如 SEQ ID No.l和 SEQ ID No.2所 示。
3、 如权利要求 2所述的抗体， 其特征在于, 其为单链抗体 ScFv 或全抗体免疫球蛋白 IgG。
4、 权利要求 1-3任一项所述的抗体经过改造得到的衍生抗体， 所述改造包括氨基酸的缺失、 替换和 /或插入， 并且不改变抗体的活 性。
5、 编码权利要求 1-4任一项所述的抗体的基因。
6、 如权利要求 5所述的基因， 其特征在于， 编码轻链可变区的 核苷酸序列和编码重链可变区的核苷酸序列如 SEQ ID No.3和 SEQ ID No.4所示。
7、 含有权利要求 5或 6所述基因的表达载体。
8、 含有权利要求 7所述表达载体的宿主。
9、 权利要求 1-4任一项所述的抗体在制备预防或治疗狂犬病的 药物中的应用。
10、 含有权利要求 1-4任一项所述的抗体的药物或检测试剂。
PCT/CN2010/001574 2010-05-06 2010-10-09 人源抗狂犬病毒糖蛋白中和性抗体rvfab3 WO2011137569A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201010171612.9 2010-05-06
CN2010101716129A CN101812132B (zh) 2010-05-06 2010-05-06 人源抗狂犬病毒糖蛋白中和性抗体(RVFab3)

Publications (1)

Publication Number Publication Date
WO2011137569A1 true WO2011137569A1 (zh) 2011-11-10

Family

ID=42619510

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2010/001574 WO2011137569A1 (zh) 2010-05-06 2010-10-09 人源抗狂犬病毒糖蛋白中和性抗体rvfab3

Country Status (2)

Country Link
CN (1) CN101812132B (zh)
WO (1) WO2011137569A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013174003A1 (en) * 2012-05-24 2013-11-28 Mountgate Group Limited Compositions and methods related to prevention and treatment of rabies infection
CN114957458A (zh) * 2021-05-08 2022-08-30 南昌大学 一种高亲和力抗狂犬病病毒的全人源单克隆抗体及其用途

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101812132B (zh) * 2010-05-06 2012-07-04 中国疾病预防控制中心病毒病预防控制所 人源抗狂犬病毒糖蛋白中和性抗体(RVFab3)
CN113501873B (zh) * 2021-07-07 2023-05-23 高光 Rbv的蛋白结合分子及其用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1355253A (zh) * 2000-11-29 2002-06-26 中国预防医学科学院病毒学研究所 人源抗狂犬病毒中和性基因工程抗体
WO2005000225A2 (en) * 2003-06-02 2005-01-06 University Of Oxford Production of rabies antibodies in plants
CN101696242A (zh) * 2009-10-26 2010-04-21 中国人民解放军南京军区军事医学研究所 人源抗狂犬病毒中和抗体Fab及其制备方法和应用
CN101812132A (zh) * 2010-05-06 2010-08-25 中国疾病预防控制中心病毒病预防控制所 人源抗狂犬病毒糖蛋白中和性抗体(RVFab3)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101550189B (zh) * 2008-04-25 2011-10-19 中国人民解放军军事医学科学院卫生学环境医学研究所 人源抗狂犬病毒糖蛋白中和性基因工程抗体rd9及其制备与应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1355253A (zh) * 2000-11-29 2002-06-26 中国预防医学科学院病毒学研究所 人源抗狂犬病毒中和性基因工程抗体
WO2005000225A2 (en) * 2003-06-02 2005-01-06 University Of Oxford Production of rabies antibodies in plants
CN101696242A (zh) * 2009-10-26 2010-04-21 中国人民解放军南京军区军事医学研究所 人源抗狂犬病毒中和抗体Fab及其制备方法和应用
CN101812132A (zh) * 2010-05-06 2010-08-25 中国疾病预防控制中心病毒病预防控制所 人源抗狂犬病毒糖蛋白中和性抗体(RVFab3)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG SHI-ZHEN ET AL.: "Generation of Human Monoclonal Antibody Fab Fragment to Rabies Virus by Phage Display Technology", CHINESE JOURNAL OF VIROLOGY, vol. 17, no. 1, March 2001 (2001-03-01), pages 11 - 16 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013174003A1 (en) * 2012-05-24 2013-11-28 Mountgate Group Limited Compositions and methods related to prevention and treatment of rabies infection
EP2855521A1 (en) * 2012-05-24 2015-04-08 Mountgate Group Limited Compositions and methods related to prevention and treatment of rabies infection
EP2855521A4 (en) * 2012-05-24 2016-03-02 Mountgate Group Ltd COMPOSITIONS AND METHODS ASSOCIATED WITH THE PREVENTION AND TREATMENT OF RABIC INFECTION
US9290564B2 (en) 2012-05-24 2016-03-22 Mountgate Group Limited Compositions and methods related to the prevention and treatment of rabies infection
EP3508497A1 (en) * 2012-05-24 2019-07-10 Mountgate Group Limited Compositions and methods related to prevention and treatment of rabies infection
CN114957458A (zh) * 2021-05-08 2022-08-30 南昌大学 一种高亲和力抗狂犬病病毒的全人源单克隆抗体及其用途
CN114957458B (zh) * 2021-05-08 2023-10-31 南昌大学 一种高亲和力抗狂犬病病毒的全人源单克隆抗体及其用途

Also Published As

Publication number Publication date
CN101812132B (zh) 2012-07-04
CN101812132A (zh) 2010-08-25

Similar Documents

Publication Publication Date Title
CN111620945B (zh) 一种抗新型冠状病毒的单克隆抗体或其衍生体
WO2011137570A1 (zh) 人源抗狂犬病毒糖蛋白中和性抗体rvfab8
CN111560070B (zh) 针对新型冠状病毒np蛋白的抗体及其检测应用
CA2809780C (en) Influenza virus neutralizing antibody and method for screening same
US20210171611A1 (en) Antibody that binds to envelope glycoprotein of sever fever with thrombocytopenia syndrome virus and use for same
CN110317267B (zh) 针对狂犬病病毒的双特异性抗体及其用途
CN116063464A (zh) 冠状病毒的抗体或其抗原结合片段
JP2019507103A (ja) 抗呼吸器合胞体ウイルスの完全ヒト抗体
WO2011137571A1 (zh) 人源抗狂犬病毒糖蛋白中和性抗体rvfab5
JP6055763B2 (ja) ヒトサイトメガロウイルス（ＣＭＶ）ｇＢタンパク質に対する高親和性ヒト抗体
JP2023510787A (ja) 抗ａｎｇｐｔｌ３抗体及びその応用
WO2010139196A1 (zh) 人源抗人干扰素α抗体及其应用
CN112574300A (zh) 抗sar-cov-2全人源单克隆抗体及其制法与应用
WO2011137569A1 (zh) 人源抗狂犬病毒糖蛋白中和性抗体rvfab3
Tsui et al. Isolation of a neutralizing human RSV antibody from a dominant, non-neutralizing immune repertoire by epitope-blocked panning.
Tikunova et al. The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library
JP2014526886A (ja) マクロファージ遊走阻止因子（ｍｉｆ）とｄ−ドーパクロームトートメラーゼ（ｄ−ｄｔ）に交差反応性がある抗体
WO2021238854A1 (zh) 抗SARS-CoV-2刺突蛋白的单克隆抗体及其制备方法和用途
WO2021233433A1 (zh) 抗SARS-CoV-2刺突蛋白单克隆抗体
WO2023046039A1 (zh) 抗SARS-CoV-2 E484Q刺突蛋白的单克隆抗体及其应用
US20230279077A1 (en) Anti-glycoprotein d antibodies, methods of preparation, and uses thereof
CN105859882B (zh) 人源抗mers病毒中和性抗体a1、其制备方法及应用
Guo et al. Construction of immune human library and screening of neutralizing antibodies specific to glycoprotein of severe fever with thrombocytopenia syndrome virus
Zhou et al. RETRACTED: A neutralizing scFv antibody against infectious bursal disease virus screened by flow cytometry
CN115724954A (zh) 抗SARS-CoV-2病毒的单克隆抗体6G21H及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10850939

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10850939

Country of ref document: EP

Kind code of ref document: A1