WO2020200143A1 - 分泌抗狂犬病毒m蛋白单克隆抗体的杂交瘤细胞株及应用 - Google Patents

分泌抗狂犬病毒m蛋白单克隆抗体的杂交瘤细胞株及应用 Download PDF

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WO2020200143A1
WO2020200143A1 PCT/CN2020/081974 CN2020081974W WO2020200143A1 WO 2020200143 A1 WO2020200143 A1 WO 2020200143A1 CN 2020081974 W CN2020081974 W CN 2020081974W WO 2020200143 A1 WO2020200143 A1 WO 2020200143A1
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monoclonal antibody
rabv
protein
rabies virus
hybridoma cell
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周继勇
廖敏
颜焰
胡伯里
顾金燕
郑肖娟
董伟仁
金玉兰
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Zhejiang University
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Zhejiang University
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • G01N2333/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus

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  • the present invention relates to the field of biotechnology, in particular to a hybridoma cell line secreting monoclonal antibodies against rabies virus M protein and applications.
  • Rabies (Rabies) is a highly lethal zoonotic disease caused by rabies virus (RABV). After the onset of RABV infection, the fatality rate is almost 100%, which is by far the acute infectious disease with the highest mortality in human history. At present, there is still no treatment, only the prevention of RABV infection through vaccination. Rabies cases have been reported in more than 150 countries around the world, and about 50,000 to 70,000 people die from rabies every year, 95% of which occur in Africa and Asia. The number of human rabies infections in China is second only to India and second in Asia. Dogs are the most common virus reservoir for RABV, and 99% of human infections with rabies are mediated by canine transmission.
  • RABV belongs to the Rhabdoviridae (Rhabdoviridae) rabies virus genus (Lyssavirus) in virus taxonomy. It is a single-stranded non-segmented RNA virus. Its genome mainly encodes nucleoprotein (N), phosphoprotein (P), Matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase protein (L). M protein, composed of 202 amino acids and about 23kDa, is the smallest and most abundant non-glycosylated protein of RABV virus particles. It is located between the viral envelope and the nucleocapsid and serves as a connection between the G protein and the nucleocapsid. The role of the shell.
  • M protein has a variety of functions: it participates in the assembly and budding of virus particles, plays a decisive factor in the shape of virus particles, regulates the transcription and translation process of the virus, participates in determining the pathogenicity of the virus, induces apoptosis, and inhibits The expression of host-related genes, etc.
  • the monoclonal antibodies against RABV protein that have been prepared are mainly NP protein and G protein.
  • NP protein monoclonal antibody can be used to distinguish different serotypes of rabies virus;
  • G protein monoclonal antibody shows higher neutralizing activity.
  • the lack of understanding of the biological characteristics of these monoclonal antibodies also limits the anti-M protein monoclonal antibodies in viruses Application in diagnosis, and research on the structure and antigenic properties of M protein.
  • the purpose of the present invention is to provide a hybridoma cell line that can secrete a monoclonal monomer that specifically recognizes RABV M protein.
  • the monoclonal antibody secreted can specifically react with the RABV strain, but not with the vaccine Flury strain. reaction.
  • hybridoma cell line that secretes a monoclonal antibody against the M protein of rabies virus.
  • the hybridoma cell line is classified and named as hybridoma cell line 4A1, which was deposited in the China Type Culture Collection on April 1, 2019, address : Wuhan University, Wuhan, China, the deposit number is CCTCC NO: C201947.
  • the present invention firstly obtains 12 strains of hybridoma cells stably secreting monoclonal antibodies against rabies virus RABV M protein from the established hybridoma cell bank, and finally screens out the hybridoma cell 4A1 strain, which has excellent biological characteristics.
  • the titer of the prepared mouse ascites monoclonal antibody 4A1 was 1 ⁇ 10 5 by indirect enzyme-linked immunosorbent assay.
  • the invention also provides a monoclonal antibody against the M protein of rabies virus prepared by the hybridoma cell 4A1 strain.
  • the present invention identifies the variant antigen epitope recognized by the monoclonal antibody for the first time, and its amino acid sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • This epitope is relatively conserved on the M protein of different RABV strains, but there is an antigenic variation on the vaccine Flury strain (the amino acid sequence of the Flury strain is shown in SEQ ID NO: 3), resulting in the monoclonal antibody not being able to recognize it. Therefore, the monoclonal antibody provided by the present invention can react specifically with the rabies virus strain, but cannot react with the vaccine Flury strain.
  • the invention provides the application of the hybridoma cell 4A1 strain in preparing a kit for detecting rabies virus RABV.
  • the hybridoma cell 4A1 strain can be used in methods such as ELISA, immunofluorescence, WesternBlot, immunohistochemistry, etc., and can be used in the preparation of reagents for clinical diagnosis of RABV infection, or in the detection of the pathogen of RABV that is not for the purpose of diagnosis .
  • the invention provides the application of the monoclonal antibody in preparing a kit for detecting rabies virus RABV.
  • the monoclonal antibody can be applied to methods such as ELISA, immunofluorescence, Western Blot, immunohistochemistry, etc., and can be used in preparing reagents for clinical diagnosis of RABV infection, or in detecting the pathogen of RABV that is not for the purpose of diagnosis.
  • the present invention also provides a kit for detecting rabies virus RABV, which includes the monoclonal antibody against the M protein of rabies virus.
  • the hybridoma cell 4A1 strain provided by the present invention has excellent biological characteristics, and the monoclonal antibody 4A1 secreted by it can specifically react with the RABV strain, but cannot react with the Flury strain; the monoclonal antibody provided by the present invention can be used for preparation ELISA, immunofluorescence, Western Blot, immunohistochemistry and other methods of kits can be used for the clinical detection of RABV or the differential diagnosis of Flury strains and other strains.
  • Figure 1 shows the specificity experiment of monoclonal antibodies.
  • Figure 2 shows the comparison of the epitopes recognized by monoclonal antibodies in different RABV strains.
  • Figure 3 shows the reactivity of monoclonal antibodies with different strains.
  • Figure 4 shows the reactivity of monoclonal antibodies with different mutant epitopes.
  • Figure 5 shows the immunohistochemistry experiment of monoclonal antibodies.
  • M protein gene (serial number: Design specific primers for the nucleotide sequence of ADJ29910.1), use the extracted viral RNA reverse transcription product as a template to amplify the M protein gene, and then clone the M protein gene into the pET-28a(+) vector and sequence
  • the competent cell E.coli BL21(DE3) was transformed, and after induction with IPTG (1mM) at 37°C for about 5h, the recombinant M protein was purified with Ni-NTA column (purchased from QiAGEN).
  • the expression and purification of recombinant M protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western RNA RNA RNA reverse transcription product.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • mice purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.
  • 100 ⁇ g/mouse were subcutaneously injected into the back.
  • an equal volume of Freund's complete adjuvant purchased from Sigma
  • Freund's incomplete adjuvant purchased from Sigma
  • blood was collected from the tail and the serum antibody titer was determined.
  • PEG cell fusion method Take myeloma cells SP2/0 and immunized BALB/c mouse spleen cells at a ratio of 1:10 and mix thoroughly, and add 1 mL of 50% PEG-4000 (purchased from Sigma) preheated to 37°C within 1 min , Gently shake while adding, finish adding within 1min, and then stand still for 1min. Add serum-free RPMI-1640 (purchased from Hyclone) medium preheated to 37°C along the tube wall to terminate the fusion reaction. It is required to add 4mL in the first minute, add 1mL in the first 30s, and 3mL in the next 30s; add 11mL in the second minute, and add to the total volume of 40mL in the last 3 minutes.
  • each well was then determined by comparing the OD values of 450 nm, P OD of 450 nm for the value of each test sample wells, N 450nm value of the negative reference serum OD, the N ⁇ 0.1, the reference positive serum OD ⁇ 2,1 450nm ratio value OD 450nm value of the negative reference sera, i.e. the female, the premise of the positive control was established, the detection hole determination P / N ⁇ 2.1 is positive, the detection hole 2.1> P / N ⁇ 1.5 hybridomas The cell line was judged to be suspicious, and the test hole with P/N ⁇ 1.5 was judged to be negative. Test again after 3 days, and select the hybridoma cell lines with positive results for both times for cloning.
  • mice were intraperitoneally injected with hybridoma cell lines (0.5mL/each, containing 2 ⁇ 10 6 ⁇ 5 ⁇ 10 6 hybridoma cells), 7 ⁇ 10 days later, take the ascites from mice with obvious bulging abdomen, centrifuge at 5000rpm for 10min, collect the supernatant, it is RABV monoclonal antibody , After aliquoting, store at -20°C for later use.
  • Hybridoma cell line 4A1 was deposited in the China Type Culture Collection on April 1, 2019, address: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: C201947, classification designation: Hybridoma cell line 4A1.
  • the subclass determination was carried out in accordance with the operating instructions of the monoclonal antibody subclass identification kit (purchased from Thermo). The results showed that the subtype of monoclonal antibody 4A1 was IgG2b ⁇ .
  • the monoclonal antibody-secreting hybridoma cell line 4A1 was cultured continuously for 20 times, cryopreserved in liquid nitrogen, and resuscitated. Purified prokaryotic expression of RABV M protein was used as the detection antigen, and the monoclonal antibody 4A1 was continuously detected by the indirect ELISA method. As a result, the ELISA titer of antibodies secreted by hybridoma cells of each generation is basically the same, reaching 10 3 -10 4 .
  • Indirect immunofluorescence test was used to verify the reactivity of monoclonal antibody 4A1 with RABV.
  • Infect BHK-21 cells purchased from ATCC with CVS-11 strain, aspirate the cell culture medium after culturing for 48 hours, add methanol-acetone (1:1) fixative solution pre-cooled at -20°C, 200uL/well, -20 After fixing at °C for 15 minutes, discard the fixative solution, wash 3 times with PBS and pat dry; add 5% skimmed milk powder (200uL/well), block at 37°C for 1 hour, wash 3 times with PBST, add monoclonal antibody 4A1 (1:500 ), incubate at 37°C for 1h, wash 3 times with PBS, pat dry, add FITC-labeled goat anti-mouse IgG antibody (purchased from KPL) diluted 1:500, incubate at 37°C for 1h, wash 3 times with PBS, pat dry each well Add 50uL PBS and observe under a fluor
  • primers were designed gradually from the N-terminus and C-terminus of the preliminarily positioned peptides in units of 1 amino acid residue, and BamH I/Xho I restriction sites were introduced upstream and downstream respectively (Table 2 ), the target fragment is obtained by annealing the upstream and downstream primers, and the target fragment is cloned into the expression vector pGEX-4T-1 to construct a recombinant plasmid, transformed into competent cells E.coli BL21(DE3), and detected by Western blot for the induced recombinant GST The reactivity of the polypeptide with the monoclonal antibody determines that the epitope recognized by the monoclonal antibody is 25 PPYDDD 30 .
  • the identified epitope sequence 25 PPYDDD 30 was compared with the amino acid sequence of the M protein of the RABV strain registered by NCBI ( Figure 2). The results showed that the epitope was mainly 25 PPDDDD 30 sequence in the RABV strain. After the mutations of amino acids P26L and Y27D were detected by Western blot, it was found that after the mutations of P26L and Y27D, the monoclonal antibody can still react with them, indicating that the monoclonal antibody 4A1 recognizes the epitope of the CVS-11 strain by Y27D substitution. But it does not affect the antigenicity of the epitope ( Figure 4).
  • Intracranial injection of 6-8 weeks old C57BL/6 mice (purchased from Shanghai Xipuer-Bikai Laboratory Animal Co., Ltd.) 30uL CVS-11 strain (10 5 TCID 50 /mL) and HEP-Flury strain, wait When the symptoms of the mice are obvious, the mice are anesthetized with 2.5% avertin (purchased from sigma) and the brains are taken. An equal volume of DMEM was injected intracranially as a blank control.
  • Antigen retrieval place the tissue section in a retrieval box filled with citric acid antigen retrieval buffer (pH 6.0) (purchased from Fuzhou Maixin Biotechnology Development Company) in a microwave oven at medium fire for 5 minutes, stop fire for 5 minutes, and turn to low Fire for 10 minutes for antigen retrieval. During this process, prevent excessive evaporation of the buffer, and do not dry the film. After natural cooling, the slides were washed 3 times in PBS (pH 7.4), 5 min each time.
  • citric acid antigen retrieval buffer pH 6.0
  • PBS pH 7.4
  • Block endogenous peroxidase put the slices into 3% hydrogen peroxide solution, incubate at room temperature for 25 minutes in the dark, and wash the slides in PBS (pH 7.4) for 3 times, each time for 5 minutes.
  • Serum blocking 3% BSA (purchased from Servicebio) was dripped into the histochemistry circle to evenly cover the tissue, and sealed at room temperature for 30 minutes.
  • DAB color development the slides were washed 3 times in PBS (pH 7.4) for 5 min each time. After the slices were dried slightly, freshly prepared DAB chromogenic solution (purchased from Fuzhou Maixin Biotechnology Development Company) was dripped into the circle, and the color development time was controlled under the microscope, and the positive color was brownish yellow. The slices were washed with tap water to stop the color development.
  • Counter-stained cell nuclei hematoxylin counter-stained for about 3 minutes, washed with tap water, differentiated with hematoxylin differentiation solution for a few seconds, washed with tap water, hematoxylin blue solution returned to blue, rinsed with running water.

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Abstract

一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株4A1，其于2019年4月1日保藏于中国典型培养物保藏中心，地址：中国武汉，武汉大学，保藏编号为CCTCC NO：C201947。通过该杂交瘤细胞株制备的单克隆抗体鉴定了其识别RABV M蛋白的精细抗原变异表位。该单克隆抗体应用于制备检测狂犬病毒RABV的试剂盒，可以检测RABV感染以及鉴别诊断疫苗Flury毒株和其它RABV毒株。

Description

分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株及应用 技术领域
本发明涉及生物技术领域，具体涉及一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株及应用。
背景技术
狂犬病(Rabies)是由狂犬病毒(RABV)引起的一种高度致死性人畜共患传染病。RABV感染发病后，致死率几乎为100％，是迄今为止人类历史上死亡率最高的急性传染病。目前仍无任何治疗措施，只能通过疫苗免疫接种预防RABV感染。在全球范围内有150多个国家均有狂犬病病例报道，每年约有5～7万人死于狂犬病，其中95％的病例发生在非洲和亚洲。中国的人狂犬病感染病例仅次于印度，位于亚洲第二位。犬是RABV最常见的病毒储藏库，99％的人感染狂犬病是由犬传播介导的。
RABV在病毒分类学上属于弹状病毒科(Rhabdoviridae)狂犬病毒属(Lyssavirus)的成员，是单股副链非节段RNA病毒，其基因组主要编码核蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)和RNA依赖的RNA聚合酶蛋白(L)。M蛋白，由202个氨基酸组成，约23kDa，是RABV病毒粒子最小、含量最丰富的一种非糖基化蛋白，其位于病毒囊膜和核衣壳之间，起到连接G蛋白和核衣壳的作用。M蛋白具有多种功能：参与病毒粒子的组装和出芽，对病毒粒子形态呈长杆状起到决定因素，调控病毒的转录和翻译过程，参与决定病毒的致病力，诱导细胞凋亡，抑制宿主相关基因的表达等。
目前已制备出的抗RABV蛋白的单克隆抗体主要是NP蛋白和G蛋白。NP蛋白单克隆抗体可用来区分狂犬病毒属不同血清型；G蛋白单克隆抗体则表现出较高的中和活性。RABV M蛋白的单克隆抗体报道很少，而且M蛋白单克隆抗体识别的精细表位还不清楚，缺乏对这些单克隆抗体生物学特性的了解，因此也限制了抗M蛋白单克隆抗体在病毒诊断中的应用，以及对M蛋白结构和抗原特性的研究。
发明内容
本发明的目的在于提供一种能分泌特异性识别RABV M蛋白的单克隆单体的杂交瘤细胞株，其分泌的单克隆抗体能与RABV毒株发生特异性反应，不能与疫苗Flury毒株发生反应。
为实现上述目的，本发明采用如下技术方案：
一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株的分类命名为杂交瘤细胞株4A1，于2019年4月1日保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，保藏编号为CCTCC NO：C201947。
本发明首先从建立的杂交瘤细胞库中得到12株稳定分泌抗狂犬病毒RABV M蛋白单克隆抗体的杂交瘤细胞，并最终筛选出杂交瘤细胞4A1株，其生物学特性优良。采用间接酶联免疫吸附分析法测得制备的小鼠腹水单克隆抗体4A1的效价为1×10 5。
本发明还提供了由杂交瘤细胞4A1株制备得到的抗狂犬病毒M蛋白的单克隆抗体。
本发明首次鉴定了该单克隆抗体所识别的变异抗原表位，其氨基酸序列如SEQ ID NO:1和SEQ ID NO:2所示。该抗原表位在不同的RABV毒株M蛋白上相对保守，但在疫苗Flury毒株上存在抗原变异(Flury毒株氨基酸序列如SEQ ID NO:3所示)，导致单克隆抗体不能识别。因此，本发明提供的单克隆抗体能与狂犬病毒毒株发生特异性反应，但是不能与疫苗Flury毒株反应。
本发明提供了杂交瘤细胞4A1株在制备检测狂犬病毒RABV的试剂盒中的应用。所述杂交瘤细胞4A1株可以应用于ELISA、免疫荧光、WesternBlot、免疫组化等方法，可以在制备临床诊断RABV感染试剂中得到应用，或在不以诊断为目的的RABV的病原检测中得到应用。
本发明提供了所述的单克隆抗体在制备检测狂犬病毒RABV的试剂盒中的应用。所述单克隆抗体可以应用于ELISA、免疫荧光、Western Blot、免疫组化等方法，可以在制备临床诊断RABV感染试剂中得到应用，或在不以诊断为目的的RABV的病原检测中得到应用。
本发明还提供了一种检测狂犬病毒RABV的试剂盒，包括所述的抗狂 犬病毒M蛋白的单克隆抗体。
本发明具备的有益效果：
本发明提供的杂交瘤细胞4A1株生物学特性优良，其分泌的单克隆抗体4A1能与RABV毒株发生特异性反应，但是不能与Flury毒株反应；本发明提供的单克隆抗体可应用于制备ELISA、免疫荧光、Western Blot、免疫组化等方法的试剂盒，可用于RABV的临床检测或者是Flury毒株和其他毒株的鉴别诊断。
附图说明
图1为单克隆抗体的特异性实验。
图2为单克隆抗体识别表位在不同RABV毒株的比对。
图3为单克隆抗体与不同毒株的反应性。
图4为单克隆抗体与不同突变表位的反应性。
图5为单克隆抗体的免疫组化实验。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1
(一)分泌单克隆抗体杂交瘤细胞株的建立
1.CVS-11M蛋白的原核表达及纯化
根据GenBank中注册的CVS-11株(Liu Juan,et al.BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein.Autophagy,2017,13(4):739-753)M蛋白基因(序列号：ADJ29910.1)的核苷酸序列设计特异引物，以提取的病毒RNA反转录产物为模板，进行M蛋白基因的扩增，然后将M蛋白的基因克隆至pET-28a(+)载体，测序验证正确后转化感受态细胞E.coli BL21(DE3)，在37℃条件下用IPTG(1mM)诱导约5h后，用Ni-NTA柱(购自QiAGEN)纯化重组M蛋白。重组M蛋白的表达和纯化用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western blot)分析。
2.动物免疫
用以上纯化的M蛋白作为免疫原，背部皮下注射免疫6～8周龄雌性 BALB/c小鼠(购自上海西普尔-必凯实验动物有限公司)，100μg/只。首免用等体积弗氏完全佐剂(购自Sigma)乳化抗原，以后每隔14d用弗氏不完全佐剂(购自Sigma)乳化抗原，加强免疫2次。第3次免疫一周后断尾采血，测定小鼠血清抗体效价。选取血清抗体效价>10 6的小鼠，细胞融合前3d用不加佐剂的抗原以腹腔注射的方式再次加强免疫(200μg/只)。
3.细胞融合
采用PEG细胞融合方法。取骨髓瘤细胞SP2/0与完成免疫的BALB/c小鼠脾细胞按1:10的比例充分混匀，在1min内加完预热至37℃的50％PEG-4000(购自Sigma)1mL，边加边轻轻振荡，1min内加完，再静置1min。沿管壁加入已预热至37℃的无血清RPMI-1640(购自Hyclone)培养基，终止融合反应。要求在第1min内加完4mL，前30s内加入1mL，后30s内加入3mL；在第2min内加完11mL，最后3min内加至总体积40mL。然后1000rpm低速离心5min，弃去上清，再沿管壁加入30mL RPMI-1640不完全培养基，再次离心、弃上清，以尽可能地去除残留的融合剂PEG。沿50mL离心管壁加入40mL预热至37℃的含HAT(购自Sigma)和10％FBS(购自Gibco)完全培养基，轻柔地吹打以重悬细胞。将重悬的细胞转移至10cm细胞培养皿内，将混匀的细胞以100μL/孔接种至已铺有饲养层细胞的96孔细胞培养板中，然后将细胞培养板放置于37℃、5％CO 2的细胞培养箱(购自Thermo)中静置培养。5d后改用含HT(购自Sigma)和10％FBS(购自Gibco)的RPMI-1640(购自Hyclone)培养基，10d后换成含10％FBS(购自Gibco)的RPMI-1640(购自Hyclone)培养基培养，当融合的细胞生长至96孔板孔底1/10-1/5时，取培养上清检测抗体。
4.杂交瘤细胞的筛选
用0.05mol/L碳酸盐缓冲液(pH9.6)对纯化的M蛋白进行稀释，并以0.02mg/mL蛋白浓度包被ELISA板(100μL/孔)，置4℃包被过夜(不少于12h)，PBST洗涤3次，每次5min，第3次后拍干；以含10％脱脂奶粉的PBS进行封闭(200μL/孔)，37℃放置1h，PBST洗涤3次，每次5min，第3次后拍干；将融合后14d的细胞上清、1:1000稀释的免疫小鼠阳性血清和1:1000稀释的小鼠阴性血清分别加入相应孔内(100μL/ 孔)，37℃作用1h，PBST洗涤3次，每次5min，第3次后拍干；加入1:10000稀释的辣根过氧化物酶(HRP)标记的羊抗鼠IgG(购自KPL)的脱脂奶粉(100μL/孔)，37℃作用1h，PBST洗涤3次，每次5min，第3次后拍干；加入底物TMB-H 2O 2(100μL/孔)，37℃避光作用10min；每孔加入50μL硫酸(2mol/L)终止反应。以空白对照调零，酶标仪测定各孔的OD 450nm值，P为各待测孔中样品的OD 450nm值，N为阴性参考血清的OD 450nm值，将N≤0.1，阳性参考血清的OD 450nm值与阴性参考血清的OD 450nm值的比值≥2.1，即阴、阳性对照成立的前提下，将P/N≥2.1的检测孔判为阳性，2.1＞P/N≥1.5的检测孔杂交瘤细胞株判为可疑，P/N＜1.5的检测孔判为阴性。隔3d后再检测一次，选择两次检测结果均为阳性的杂交瘤细胞株进行克隆化。
5.杂交瘤细胞的克隆化
首先将阳性孔活细胞用台盼兰进行染色和计数，用含10％FBS(购自Gibco)的RPMI-1640培养基(购自Hyclone)稀释成100个细胞/10mL培养基的细胞悬液，将稀释的细胞悬液加入已铺有饲养层细胞的96孔细胞培养板(100μL/孔)，置于37℃、5％CO 2培养箱中培养，每天于光学显微镜下观察克隆细胞的形成情况，记录只有单个克隆的生长孔，当细胞生长至96孔板孔底1/10-1/5时取细胞上清用于ELISA检测。选择检测为阳性的单克隆细胞再进行同样的克隆，直至克隆后所有细胞孔上清检测均为阳性，取OD 450nm值最大的孔进行扩大培养，最终从杂交瘤细胞库中筛选到了12株稳定分泌RABV特异性单克隆抗体的杂交瘤细胞。
6.单克隆抗体腹水的制备
将灭菌的液体石蜡腹腔注射6～8周龄的雌性BALB/c小鼠(购自上海西普尔-必凯实验动物有限公司)，0.5mL/只，7d后小鼠腹腔注射杂交瘤细胞株(0.5mL/只，含2×10 6～5×10 6个杂交瘤细胞)，7～10d后采取腹部明显鼓起小鼠的腹水，5000rpm离心10min，收集上清，即为RABV单克隆抗体，分装后于-20℃保存备用。
(二)单克隆抗体4A1的生物学特性
生物保藏
杂交瘤细胞4A1株，于2019年4月1日保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，保藏编号为CCTCC NO：C201947， 分类命名：杂交瘤细胞株4A1。
1.单克隆抗体的类型测定
亚类测定按照单克隆抗体亚类鉴定试剂盒(购自Thermo)操作说明书进行，结果显示，单克隆抗体4A1亚型为IgG2bκ。
2.单克隆抗体效价和稳定性测定
将分泌单克隆抗体的杂交瘤细胞4A1株进行连续培养传代20次、液氮冻存与复苏试验，以纯化的原核表达的RABV M蛋白为检测抗原，通过间接ELISA方法连续检测单克隆抗体4A1的效价，结果各代次杂交瘤细胞分泌的抗体ELISA效价基本一致，达到10 3-10 4。
3.单克隆抗体的特异性鉴定
采用间接免疫荧光试验验证单克隆抗体4A1与RABV的反应性。用CVS-11毒株感染BHK-21细胞(购自ATCC)，培养48h后吸弃细胞培养液，加入-20℃预冷的甲醇-丙酮(1:1)固定液，200uL/孔，-20℃固定15min后，弃去固定液，用PBS洗涤3次后拍干；加入5％脱脂奶粉(200uL/孔)，37℃封闭1h后，PBST洗涤3次，加入单克隆抗体4A1(1:500)，37℃孵育1h，PBS洗涤3次，拍干后加入1:500稀释的FITC标记的羊抗鼠IgG抗体(购自KPL)，37℃孵育1h，PBS洗涤3次，拍干后每孔加入50uL PBS，置于荧光显微镜下观察。单克隆抗体4A1与CVS-11感染的BHK-21细胞反应，产生绿色荧光，而正常未感染CVS-11毒株的BHK-21细胞无绿色荧光(见图1)。
4.单克隆抗体识别抗原表位的鉴定
将M蛋白基因609bp序列(aa1-aa203)分为相互重叠的片段，设计引物(表1)，上、下游分别引入Hind Ⅲ/Kpn Ⅰ酶切位点，将扩增纯化的目的片段克隆于表达载体pET-28a(+)中构建重组质粒，转化感受态细胞E.coli BL21(DE3)，诱导表达的重组His标签多肽与单克隆抗体进行Western blot验证，对阳性区段逐步进行截短表达，完成抗原表位初步定位。为精确鉴定抗原表位，从初步定位的肽段的N端和C端分别以1个氨基酸残基为单位逐步递减设计引物，上、下游分别引入BamH Ⅰ/Xho Ⅰ酶切位点(表2)，通过上、下游引物退火得到目的片段，将目的片段克隆于表达载体pGEX-4T-1中构建重组质粒，转化感受态细胞E.coli BL21(DE3)，用Western blot检测诱导表达的重组GST多肽与单克隆抗体的反 应性，确定单克隆抗体识别的抗原表位为 25PPYDDD 30。
表1鉴定单克隆抗体抗原表位所在区段引物
Figure PCTCN2020081974-appb-000001
表2鉴定单克隆抗体精细表位引物
Figure PCTCN2020081974-appb-000002
5.单克隆抗体表位序列分析
将鉴定的抗原表位序列 25PPYDDD 30与NCBI注册的RABV毒株M蛋白氨基酸序列进行比对分析(图2)，结果表明该抗原表位在RABV毒株中主要是 25PPDDDD 30序列。对氨基酸P26L和Y27D分别突变后进行Western blot检测，结果发现P26L和Y27D突变后，该单克隆抗体仍然能够与之反应，说明单克隆抗体4A1识别CVS-11毒株的抗原表位发生Y27D置换，但是并不影响表位的抗原性(图4)。同时，Western blot实验结果发现本发明的M蛋白单克隆抗体可与CVS-11、WT型毒株反应，不能与HEP-Flury毒株(购自中国兽医微生物菌种保藏管理中心，保藏号：CVCC AV2013)反应(图3)，同时RABV毒株的N蛋白单克隆抗体可分别与3个毒株发生特性反应，说明病毒感染细胞是成功的。RABV序列比对发现HEP-Flury毒株的抗原表位序列是 25PPDGDD 30，进一步将 25PPYDDD 30表位进行氨基酸D28G突变，Western blot实验发现单克隆抗体4A1无法与之识别(图4)。然而本发明鉴定的抗原表位与Flury及变体毒株(图2：HEP-Flury、rHEP5.0-CVSG、LEP-Flury、Flury-LEP-C)发生氨基酸D28G突变，影响了表位的抗原性，因此本发明的单克隆抗体也可用于将Flury及变体毒株与其它流行RABV毒株进行区分。
6.单克隆抗体的免疫组化实验
颅内注射感染6-8周龄C57BL/6小鼠(购自上海西普尔-必凯实验动物有限公司)30uL CVS-11毒株(10 5TCID 50/mL)和HEP-Flury毒株，待小鼠发病症状明显时，用2.5％avertin(购自sigma)麻醉小鼠，取脑。以颅内注射等体积的DMEM为空白对照。
(1)固定与包埋：4％多聚甲醛固定24h，经酒精脱水与透明，浸石蜡包埋，切片。
(2)石蜡切片脱蜡至水：依次将切片放入二甲苯Ⅰ 15min-二甲苯Ⅱ 15min-二甲苯III 15min-无水乙醇Ⅰ 5min-无水乙醇Ⅱ 5min-85％酒精5min-75％酒精5min-蒸馏水洗。
(3)抗原修复：组织切片置于盛满柠檬酸抗原修复缓冲液(pH6.0)(购自福州迈新生物技术开发公司)的修复盒中于微波炉内中火5min，停火5min，转低火10min进行抗原修复，此过程中应防止缓冲液过度蒸发，切勿干片。自然冷却后将玻片置于PBS(pH7.4)中洗涤3次，每次5 min。
(4)阻断内源性过氧化物酶：切片放入3％双氧水溶液，室温避光孵育25min，将玻片置于PBS(pH7.4)中洗涤3次，每次5min。
(5)血清封闭：在组化圈内滴加3％BSA(购自Servicebio)均匀覆盖组织，室温封闭30min。
(6)加一抗：轻轻甩掉封闭液，在CVS-11毒株、HEP-Flury毒株和DMEM感染脑组织切片上滴加1:500稀释的一抗(本发明单克隆抗体)，切片平放于湿盒内4℃孵育过夜(湿盒内加少量水防止抗体蒸发)。其中HEP-Flury毒株感染脑组织切片再做一个阳性对照，该切片以HEP-Flury感染小鼠的血清为一抗。
(7)加二抗：玻片置于PBS(pH7.4)中洗涤3次，每次5min。切片稍甩干后在圈内滴加HRP标记的羊抗鼠IgG(购自KPL)覆盖组织，室温孵育50min。
(8)DAB显色：玻片置于PBS(pH7.4)中洗涤3次，每次5min。切片稍甩干后在圈内滴加新鲜配制的DAB显色液(购自福州迈新生物技术开发公司)，显微镜下控制显色时间，阳性为棕黄色，自来水冲洗切片终止显色。
(9)复染细胞核：苏木素复染3min左右，自来水洗，苏木素分化液分化数秒，自来水冲洗，苏木素返蓝液返蓝，流水冲洗。
(10)脱水封片：将切片依次放入75％酒精5min-85％酒精5min--无水乙醇Ⅰ 5min-无水乙醇Ⅱ 5min-二甲苯Ⅰ 5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。
(11)显微镜镜检，图像采集分析显示苏木素染细胞核为蓝色，DAB显出的阳性表达为棕黄色，即该单克隆抗体能对感染的脑组织中的RABV病毒进行标记(图5)。

Claims (6)

  1. 一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株，其特征在于，所述杂交瘤细胞株的分类命名为杂交瘤细胞株4A1，其保藏编号为CCTCC NO：C201947。
  2. 如权利要求1所述的杂交瘤细胞株在制备检测狂犬病毒RABV的试剂盒中的应用。
  3. 一种抗狂犬病毒M蛋白的单克隆抗体，其特征在于，所述单克隆抗体由权利要求1所述的杂交瘤细胞株制备得到。
  4. 如权利要求3所述的抗狂犬病毒M蛋白的单克隆抗体，其特征在于，所述单克隆抗体识别的狂犬病毒RABV抗原表位的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2所示。
  5. 如权利要求3所述的单克隆抗体在制备检测狂犬病毒RABV的试剂盒中的应用。
  6. 一种检测狂犬病毒RABV的试剂盒，其特征在于，包括如权利要求3所述的抗狂犬病毒M蛋白的单克隆抗体。
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