WO2011015121A1 - 抗对虾白斑症病毒囊膜蛋白vp28独特型单克隆抗体及其制备方法 - Google Patents

抗对虾白斑症病毒囊膜蛋白vp28独特型单克隆抗体及其制备方法 Download PDF

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WO2011015121A1
WO2011015121A1 PCT/CN2010/075624 CN2010075624W WO2011015121A1 WO 2011015121 A1 WO2011015121 A1 WO 2011015121A1 CN 2010075624 W CN2010075624 W CN 2010075624W WO 2011015121 A1 WO2011015121 A1 WO 2011015121A1
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wssv
monoclonal antibody
well
envelope protein
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战文斌
韦秀梅
绳秀珍
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中国海洋大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/18011—Nimaviridae
    • C12N2710/18022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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  • the present invention relates to a monoclonal antibody (monoclonal antibody) and a preparation method thereof, specifically, against white spot syndrome virus (White spot syndrome virus, WSSV)
  • white spot syndrome virus White spot syndrome virus, WSSV
  • the unique monoclonal antibody of envelope protein VP28 (virus protein 28) and its preparation method belong to the field of molecular immunology and virology cross-technology. Background technique
  • WSSV is a double-stranded DNA virus with a capsule that has high infectivity and replication capacity and a wide range of hosts.
  • Existing studies at home and abroad involve the morphological structure, chemical composition and physicochemical properties of WSSV, genomes and their encoded polypeptides, hosts and transmission pathways, and epidemiology, to identify the molecular mechanisms of WSSV infection, to find prevention and control of WSSV infection.
  • Effective methods and measures are the hotspots of current research.
  • One of the pathways by which a virus invades a cell is that the viral envelope protein binds to a host-specific cellular receptor, which in turn invades and infects the cell.
  • Anti-idiotypic antibodies have the properties of mimicking antigens and are used in medicine for receptor research, pathogenesis of autoimmune diseases, and novel vaccine research. However, there are few applications in aquatic animal diseases, about WSSV or its related proteins. Studies on anti-idiotypic antibodies have not been reported.
  • the object of the present invention is to provide an anti-WSSV-VP28 idiotype monoclonal antibody secreted by hybridoma cells and a preparation method thereof, and to further confirm the role of WSSV-VP28 in viral infection, screening for adhesion proteins of WSSV and The identification of cell receptor proteins provides new research ideas and research methods, and further studies the infection mechanism of WSSV, and achieves the purpose of preventing and controlling WSSV disease by cutting off the viral infection pathway.
  • An anti-WS SV-VP28 idiotype monoclonal antibody (Ab2) was developed, which is deposited by the China National Culture Collection, and the preservation date is: 2009 On May 11th, the accession number is: CCTCC-C200938 is secreted by the hybridoma cell AMVP.
  • the monoclonal antibody is prepared by anti-WSSV-VP28 monoclonal antibody (Ab l ) and can bind to rabbit anti-WSSV-VP28 antibody.
  • a method for preparing an anti-WSSV-VP28 idiotype monoclonal antibody which comprises: purifying a neutralizing anti-WSSV-VP28 monoclonal antibody (Ab l ) which has been verified by in vivo and in vitro neutralization experiments as an antigen, The mice were immunized, and the spleen was taken to obtain spleen cells.
  • the mouse P3-X63-Ag8Ul myeloma cells were cultured to exponential growth phase; the spleen cells were fused with myeloma cells; the fused cells were screened by immunological methods and screened.
  • Hybridoma cells secreting anti-WSSV-VP28 idiotype monoclonal antibody were cloned to obtain hybridoma cell lines by limiting dilution method; the obtained clonal cell lines were cultured by conventional methods, and the culture medium was injected or injected into the abdominal cavity of mice to prepare ascites.
  • the anti-WSSV-VP28 idiotype monoclonal antibody was obtained after purification by precipitation and affinity chromatography.
  • the immunological screening methods are indirect enzyme-linked immunosorbent assay and competitive enzyme-linked immunosorbent assay.
  • the immunological identification methods described are competitive enzyme-linked immunosorbent assay, indirect immunofluorescence assay, gold-labeled immunoelectron microscopy, and in vivo neutralization assay of crayfish.
  • the indirect enzyme-linked immunosorbent assay confirmed that the monoclonal antibody could bind to the rabbit anti-WSSV-VP28 antibody.
  • the competitive enzyme-linked immunosorbent assay confirmed that the monoclonal antibody could competitively inhibit the binding of rabbit anti-WSSV-VP28 antibody to WSSV, indirect immunofluorescence, Gold-labeled immunoelectron microscopy confirmed that both Ab3 and Ab1 could bind to the WSSV capsule, and the neutralization of the crayfish showed that Ab3 was neutralized.
  • the identification method of anti-WSSV-VP28 idiotype monoclonal antibody is: immunization of mice with purified anti-WSSV-VP28 idiotype monoclonal antibody (Ab2) to prepare anti-WSSV-VP28 idiotype antibody (Ab3), identified by immunological identification method
  • the antigenic determinant corresponding to Ab3 is located on the envelope of WSSV, and the neutralizing experiment of crayfish demonstrates that Ab3 is neutralized, indicating that Ab3 has Ab l characteristics, which proves that Ab2 has simulated WSSV-
  • the VP28 is an anti-WSSV-VP28 unique monoclonal antibody.
  • the monoclonal antibody of the present invention proves that it can compete with WSSV for binding to rabbit anti-WSSV-VP28 antibody by competitive enzyme-linked immunosorbent assay.
  • Ab3 can be specifically bound to WSSV by indirect immunofluorescence and gold-labeled immunoelectron microscopy, and its binding site is located. On the WSSV capsule, the neutralization of crayfish in vivo demonstrated that Ab3 partially neutralized WSSV infection and delayed the death of crawfish.
  • the hybridoma cells with good growth state have a full appearance, roundedness, strong refractive index, uniform cell size, and good adherence; the hybridoma cells have infinite proliferative ability; the hybridoma cells have good growth potential.
  • Conventional culture for 2-3 days the medium is changed from pink to yellow, and the medium contains the anti-proliferation of the hybridoma cells.
  • the antigenic determinant of the monoclonal antibody of the present invention is the variable region of Ab l , and Ab l has the property of neutralizing WSSV infection.
  • the invention firstly applies the anti-idiotype antibody to the WSSV research, and establishes a screening system using indirect enzyme-linked immunosorbent assay and competitive enzyme-linked immunosorbent assay, and also uses indirect immunofluorescence, gold-labeled immunoelectron microscopy and In vivo neutralization experiments of crayfish demonstrated that Ab3 has Ab l characteristics, thereby verifying that the mAb of the present invention has the property of mimicking the original antigen WSSV-VP28.
  • Fig. 1 is a diagram showing the results of transfer immunoblotting detection of Ab l .
  • Figure 2 is a graph showing the results of neutralization experiments of the crawfish in Ab l.
  • Figure 3 is a graph showing the results of a competitive enzyme-linked immunosorbent assay for monoclonal antibodies of the present invention.
  • Figure 4 is a graph showing the results of a competitive enzyme-linked immunosorbent assay for monoclonal antibody and Ab3 of the present invention.
  • Figure 5 a is a graph of indirect immunofluorescence assay of Abl.
  • b is a graph of indirect immunofluorescence detection of Ab3.
  • Figure 6 a is a graphical representation of the electron microscopy observation of the purified WSSV.
  • b is the gold-labeled immunoelectron micrograph of Abl.
  • c is the gold-labeled immunoelectron micrograph of Ab3.
  • Figure 7 is a graph showing the results of neutralization experiments of the crawfish in Ab3. BEST MODE FOR CARRYING OUT THE INVENTION
  • the present invention will be further described by way of specific embodiments in conjunction with the accompanying drawings.
  • the anti-WSSV-VP28 idiotype monoclonal antibody of the present invention is secreted by a hybridoma cell having the accession number: CCTCC-C200938, which has the property of simulating WSSV-VP28.
  • the method for preparing an anti-WSSV-VP28 idiotype monoclonal antibody of the present invention is as follows:
  • the hybridoma cell secreting anti-WSSV-VP28 monoclonal antibody (Abl) is prepared as follows: 1. Preparation of antigen
  • the virus band was taken, diluted 6 times with TE and centrifuged at 20000 g for 90 min; the resulting pellet was treated with PBS (137 mM NaCl, 2.7 mM KCl, 8.09 mM Na 2 HPO). 4 , 1.47mM KH 2 P0 4 , pH 7.4) Resuspend, negatively stained by electron microscope, dispensed, frozen and stored at _80 °C for use.
  • step (2) Add the WSSV purified in step (1) to an equal volume of sample buffer containing sodium dodecyl sulfate, boil in boiling water for 3-5 minutes, and then add to the electrophoresis apparatus (Mini-PROTEAN II, Bio- Rad). In the sample well, electrophoresis. The gel after electrophoresis was taken out and stained in CuCl 2 reversible staining solution (CuCl 2 23 ⁇ 40 5.11 g dissolved in 100 ml of double distilled water), and the VP28 protein band was cut out, decolorized, eluted, dialyzed, and lyophilized. The PBS was dissolved and the protein concentration was adjusted to 1 mg/ml, and 50 ⁇ M/tube was dispensed and stored at -80 °C.
  • the purified WSSV-VP28 protein was used as the antigen in step 1.
  • the Balb/c mice were immunized, and the immunization was divided into 4 times.
  • the first 2 immunization intervals were 2 weeks, the last 2 immunization intervals were 1 week, and the first 2 immunizations were intraperitoneal injection. , the last 2 immunizations for the tail vein injection.
  • the boosted, purified WSSV-VP28 protein was mixed in equal volume with Freund's incomplete adjuvant, each injection of 100 ⁇ l;
  • mice were immunized with ether anesthesia, and the spleen and thymus were aseptically removed, respectively, passed through a 100 mesh sieve, and blown with RPMI-1640 solution to form a single cell suspension;
  • the pelleted cells were resuspended in 3 ml of RPMI-1640 (containing 10% fetal bovine serum) cell culture medium preheated to 37 ° C; (8) The cell suspension was added to the thymocyte suspension prepared in step (2). , mixed uniformly and then added dropwise to a 96-well culture plate;
  • the culture plate was placed in a C0 2 incubator at 37 ° C, and the growth of the cells was observed under an inverted microscope, and the hybridoma cell culture solution was taken for detection.
  • the positive hybridoma cell line against WSSV was screened by indirect immunofluorescence.
  • Hybridoma cells detected as positive wells were cloned by limiting dilution method as follows:
  • mice 1 Anesthetize the mice with ether, take the thymus, and make a single cell suspension;
  • the cell suspension was evenly pipetted with a dropper and added dropwise to a 96-well culture plate at ⁇ per well, with an average of approximately one hybridoma cell per well;
  • the culture plate is placed in a 37 ° C C0 2 incubator;
  • hybridoma cells obtained from the positive single clone wells were re-cloned as described above to ensure positive antibody-cloning.
  • WSSV-VP28 positive hybridoma cell line
  • Step 1 The purified virus is added to the sample buffer, boiled, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the gel is taken out;
  • the nitrocellulose membrane is taken out; the nitrocellulose membrane is washed with PBS for 10 min, and then placed in a 3% bovine serum albumin solution at 37 ° C for 1 h;
  • nitrocellulose membrane was placed in an anti-WSSV antibody-positive hybridoma cell culture medium, incubated at 37 ° C for 1 h, and washed with the same method;
  • nitrocellulose membrane was placed in alkaline phosphatase-labeled goat anti-mouse Ig antibody (1:4000 dilution), incubated at 37 ° C for 1 h, and washed with 6 methods;
  • Example 1 WSSV purified by one (1) in the above Example 1 was diluted with sterile PBS 1:100, and the WSSV dilution was mixed with the positive monoclonal hybridoma cell culture medium in an equal volume; the WSSV dilution and bone marrow were used.
  • the volumetric mixture of tumor cell culture medium was used as a positive control; the myeloma cell culture medium was used as a negative control; after each group of samples was placed at room temperature for 2 hours, crayfish were injected for in vivo neutralization experiment.
  • M is the result of Coomassie blue staining after standard molecular weight protein electrophoresis
  • V is the result of Coomassie blue staining after purification of WSSV electrophoresis
  • 1 is the reaction of Abl with the transferred WSSV protein band, Abl recognition molecular weight is 28kDa Protein.
  • the positive control is the incubation of the myeloma cell culture medium with the WSSV dilution, and the injection into the crawfish, the mortality of the crawfish changes within 15 days, and the crawfish die on the first day after the injection, the 7th day.
  • the mortality rate is 100%; the negative control is to inject the myeloma cell culture into the crawfish, and the mortality of the crawfish changes within 15 days.
  • the crawfish mortality is 5%; Abl is the Abl and After WSSV incubation, the crayfish were injected into the crawfish, and the mortality of the crawfish changed within 15 days. The crawfish died on the 5th day and the mortality on the 15th day reached 100%.
  • mice were intraperitoneally injected with 0.5 ml of liquid paraffin, and 10 days later, hybridoma cells secreting anti-WSSV-VP28 monoclonal antibody (Abl) were intraperitoneally injected, and the mice were reared and closely observed. After 14 days of abdominal expansion of the mice Ascites was taken. The ascites was purified by octanoic acid-ammonium sulfate method and purified by Protein G Agarose (Amersham) to adjust the concentration of 2 mg/ml.
  • Abl anti-WSSV-VP28 monoclonal antibody
  • purified Abl was used as an antigen to immunize Balb/c mice, and the immunization was performed in 5 times.
  • the first 2 immunization intervals were 2 weeks, the last 3 immunization intervals were 1 week, and the first 2 immunizations were intraperitoneal injection.
  • the last 3 immunizations were injected into the tail vein.
  • the hybridoma cell population to be fused was detected when the cell bottom area of the 96-well culture plate was about 1/3, and the anti-WSSV-VP28 anti-idiotype monoclonal antibody-positive hybridoma cells were initially screened by indirect enzyme-linked immunosorbent assay.
  • Hybridoma cells which were detected as positive wells were cloned according to steps 1 to 5 in Example 1 (2). Two weeks later, the hybridoma cell culture medium of the single clone well was detected according to the fourth (1) step 1-8 of the present example, and the hybridoma cells of the positive single clone well were cloned again to ensure an antibody-positive monoclonal.
  • a total of 6 positive hybridoma cells (named as monoclonal antibodies A, B, C, D, E, F) were injected into the peritoneal cavity of mice to produce ascites, and were screened by competitive enzyme-linked immunosorbent assay.
  • Example 1 (1) The purified WSSV was diluted with a 1:40 dilution of the carbonate coating solution, and the WSSV dilution was added to the 96-well microtiter plate at ⁇ per well, and coated at 4 ° C overnight;
  • the selected monoclonal antibody A can compete with WSSV for binding to rabbit anti-WSSV-VP28 antibody. As the dilution factor of the antibody increases, the inhibition rate decreases. Monoclonal antibody A is secreted by a hybridoma cell having the accession number: CCTCC-C200938, which is an anti-WSSV-VP28 idiotype monoclonal antibody.
  • Example 1 Step 1 The purified WSSV was diluted with a carbonate coating solution of 1:40, added to a 96-well microtiter plate at a volume of ⁇ , and coated at 4 ° C overnight;
  • the monoclonal antibody of the present invention purified by caprylic acid-ammonium sulfate method is an antigen
  • the anti-serum (Ab3) is obtained by immunizing Balb/c mice
  • the monoclonal antibody and Ab3 of the present invention are diluted by PBS gradients of 10, 20, 40, 80, 160, respectively.
  • 320, 640, 1 280, 2 560, 5120 times the gradient dilutions of the monoclonal antibody and Ab3 of the present invention were separately mixed with the rabbit anti-WSSV-VP28 antibody at a concentration of 40 ⁇ g/ml into an experimental group, 4°. After overnight C, a mixture of ⁇ was added to each well, and incubated at 37 ° C for 1 h, and PBS was used instead of the monoclonal antibody of the present invention and Ab3 as a competition-free control;
  • Both the monoclonal antibody and Ab3 of the present invention can compete to inhibit the binding of the rabbit anti-WSSV-VP28 antibody to WSSV, and the inhibition rate decreases as the dilution factor increases, as shown in Fig. 4.
  • b is that sputum infected with WSSV disease shrimp is incubated with Ab3 and then incubated with fluorescein-labeled goat anti-mouse IgG antibody to observe yellow-green fluorescence.
  • the ruler in the figure is 50Mffl.
  • Example 1 The neutralization of the anti-WSSV-VP28 idiotype antibody (Ab3) was confirmed by the in vivo neutralization test of the crayfish according to the method described in the four (4) of Example 1.
  • One (1) purified WSSV in Example 1 was diluted with sterile PBS 1:100; the WSSV dilution was mixed with Ab3 in equal volume as the experimental group; the equal volume of WSSV and normal mouse serum was mixed as a positive control; Normal mouse serum was used as a negative control.
  • crawfish were injected for in vivo neutralization experiments. The remaining steps are the same as four (4) steps 2-3 in Example 1.
  • the results of the neutralization test of the crayfish used in the present invention The negative control is to inject the normal mouse serum into the crawfish, and the mortality of the crawfish changes in 15 days, and the crawfish has no death in 15 days; the positive control is Normal mouse serum-incubated WSSV was injected into crawfish, and the mortality of crayfish changed within 15 days. Crawfish died on the first day after WSSV injection. The mortality on day 7 reached 100%. Ab3 was to be incubated with Ab3. The WSSV was injected into the crawfish, and the mortality of the crawfish changed within 15 days. Crawfish began to die on the 5th day after injection, and until the 15th day, nearly 20% of the crayfish survived, as shown in Figure 7.

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Description

说 明 书
抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体及其制备方法 技术领域 本发明涉及一种单克隆抗体 （单抗）及其制备方法， 具体是抗对虾白斑症病毒 （White spot syndrome virus, WSSV) 囊膜蛋白 VP28 ( virus protein 28 ) 的独特型单抗及其制备 方法， 属于分子免疫学和病毒学交叉技术领域。 背景技术
WSSV是一种具有囊膜的双链 DNA病毒，具有很高的侵染能力和复制能力，宿主范围十分 广泛。 国内外已有的研究涉及 WSSV的形态结构、化学组成和理化特性、基因组及其编码多肽、 宿主和传播途径及流行病学等方面， 探明 WSSV感染的分子机理、 寻找预防和控制 WSSV感染的 有效方法和措施是目前研究的热点。 病毒入侵细胞的路径之一是病毒囊膜蛋白与宿主特异性 细胞受体相结合， 继而侵入并感染细胞， 因此， 研究 WSSV吸附和侵入宿主细胞过程中起关键 作用的蛋白以及宿主细胞上的病毒特异性受体蛋白， 对研究 WSSV感染的机理和预防控制具有 重要意义。 目前研究表明， 在已发现和鉴定的 WSSV蛋白中， 囊膜蛋白 VP28的特异性抗体能中 和 WSSV的感染， 因此 WSSV-VP28被认为在病毒感染过程中起着非常重要的作用。抗独特型抗体 具有模拟抗原的特性， 在医学上被用于受体研究、 自身免疫病发病机理研究和新型疫苗研究 等方面， 而在水产动物疾病研究上应用甚少， 关于 WSSV或其相关蛋白的抗独特型抗体的研究 未见报道。 发明内容 本发明的目的是提供一种由杂交瘤细胞分泌的抗 WSSV-VP28独特型单抗及其制备方法， 为进一步确认 WSSV-VP28在病毒感染中所起的作用， 筛选 WSSV的黏附蛋白和鉴定细胞受体 蛋白提供新的研究思路和研究方法， 进而深入研究 WSSV的感染机理， 通过切断病毒的感染 途径， 实现预防控制对虾 WSSV病的目的。
本发明的目的是由以下技术方案实现的： 研制了一种抗 WS SV-VP28 独特型单抗 (Ab2), 该单抗是由保藏单位为： 中国典型培养物保藏中心， 保藏日期为： 2009年 5月 11 日， 保藏号为： CCTCC— C200938的杂交瘤细胞 AMVP分泌的， 该单抗是以抗 WSSV-VP28 单抗 （Ab l ) 为抗原制备的， 能与兔抗 WSSV-VP28 抗体结合； 具有与 WSSV 竞争结合兔抗 WSSV-VP28抗体的能力； 且以该单抗为抗原制备的抗抗 WSSV-VP28独特型抗体 （Ab3 ) 能与 WSSV 结合， 其结合位点在 WSSV 的囊膜上， Ab3 能中和 WSSV 感染， 具有 Ab l 特性。 一种制备抗 WSSV-VP28独特型单抗的方法， 所述的制备方法是： 纯化经体内、体外中和实 验已验证的具有中和性的抗 WSSV-VP28单抗（Ab l )为抗原， 免疫小鼠， 取脾脏以获得脾细胞； 取小鼠 P3-X63-Ag8Ul骨髓瘤细胞，培养至指数生长期；将脾细胞与骨髓瘤细胞进行细胞融合； 融合细胞采用免疫学方法筛选，筛选得到分泌抗 WSSV-VP28独特型单抗的杂交瘤细胞，经有限 稀释法克隆得到杂交瘤细胞株； 以常规方法培养所得到的克隆细胞株， 收集培养液或注入小 鼠腹腔制备腹水， 以硫酸铵沉淀和亲和层析方法纯化后得到抗 WSSV-VP28独特型单抗。
所述的免疫学的筛选方法是间接酶联免疫吸附法和竞争酶联免疫吸附法。 所述的免疫学 的鉴定方法是竞争酶联免疫吸附法、 间接免疫荧光法、 金标记免疫电镜法和螯虾体内中和实 验。间接酶联免疫吸附法确定了该单抗能与兔抗 WSSV-VP28抗体结合，竞争酶联免疫吸附法证 明该单抗能竞争抑制兔抗 WSSV-VP28抗体与 WSSV的结合， 间接免疫荧光法、金标记免疫电镜法 证实 Ab3与 Ab l都能与 WSSV囊膜结合， 螯虾体内中和实验证明 Ab3具有中和性。
抗 WSSV-VP28独特型单抗的鉴定方法是： 以纯化的抗 WSSV-VP28独特型单抗（Ab2 )免疫小 鼠制备抗抗 WSSV-VP28独特型抗体 （Ab3)， 采用免疫学的鉴定方法鉴定 Ab3对应的抗原决定簇 与 Ab l—样， 位于 WSSV的囊膜上， 并采用螯虾体内中和实验证明 Ab3具有中和性， 表明 Ab3具有 Ab l特性， 也就证明了 Ab2具有模拟 WSSV-VP28特性， 是抗 WSSV-VP28独特型单抗。
本发明单抗经竞争酶联免疫吸附法证明能与 WSSV竞争结合兔抗 WSSV-VP28抗体， Ab3经间 接免疫荧光法、 金标记免疫电镜法证明， 能与 WSSV特异性结合并且其结合位点位于 WSSV囊膜 上， 螯虾体内中和实验证明了 Ab3能部分中和 WSSV感染， 延缓螯虾死亡。
在倒置显微镜下观察， 生长状态良好的该杂交瘤细胞， 其外观饱满、 浑圆、 折光性强、 细胞大小均一、 贴壁良好； 该杂交瘤细胞有无限分裂增殖能力； 长势良好的该杂交瘤细胞常 规培养 2-3天， 其培养基由桃红色转变为黄色， 该培养基中含有该杂交瘤细胞分泌的抗
WSSV-VP28独特型单抗。
本发明单抗的抗原决定簇是 Ab l的可变区， Ab l有中和 WSSV感染的特性。 本发明首次将该 抗独特型抗体应用于 WSSV研究中， 并且建立了运用间接酶联免疫吸附法和竞争酶联免疫吸附 法检测的筛选体系， 还采用间接免疫荧光法、 金标记免疫电镜法和螯虾体内中和实验证明了 Ab3具有 Ab l特性，从而验证了本发明单抗具有模拟最初抗原 WSSV-VP28的特性。该单抗的研制， 对进一步确认 WSSV-VP28在 WSSV感染中所起的作用， 筛选 WSSV黏附蛋白和鉴定细胞受体蛋白， 从而深入研究 WSSV的感染机理， 切断 WSSV的感染途径具有重要的意义。 附图说明 图 1为 Ab l的转印免疫印迹检测结果图。
图 2为 Ab l的螯虾体内中和实验结果图。 图 3为本发明单抗的竞争酶联免疫吸附法检测结果图。
图 4为本发明单抗及 Ab3的竞争酶联免疫吸附法检测结果图。
图 5 a为 Abl的间接免疫荧光检测结果图。
b为 Ab3的间接免疫荧光检测结果图。
图 6 a 为提纯 WSSV的电镜观察结果图。
b为 Abl的金标记免疫电镜结果图。
c为 Ab3的金标记免疫电镜结果图。
图 7 为 Ab3的螯虾体内中和实验结果图。 具体实施方式 下面结合附图并通过具体实施例进一步说明本发明。
本发明的抗 WSSV-VP28独特型单抗是由保藏号为： CCTCC一 C200938的杂交瘤细胞分泌 的， 该单抗具有模拟 WSSV-VP28的特性。
本发明的制备抗 WSSV-VP28独特型单抗的方法如下：
实施例 1
所述的分泌抗 WSSV-VP28单抗 （Abl ) 的一株杂交瘤细胞， 其制备方法如下： 一、 抗原的制备
( 1 )病毒的提纯：取感染 WSSV濒死中国对虾的头胸部 (去除肝胰腺），剪碎后加入以 T E 缓冲液 （0.05M Tris-Hcl, O. lM NaCl, 1 mM EDTA, pH7.4) 配制的 25% (W/W)蔗糖溶液中研 磨， 研磨后的样品经 600g离心 20min，取上清； 800g离心 20min，取上清； 20000g离心 2h， 弃去上清； 沉淀以 25%蔗糖溶液重悬后， 铺于不连续蔗糖梯度上 20000g离心 2h; 离心后取 病毒区带，以 T E稀释 6倍后 20000g离心 90min；所得沉淀用 PBS ( 137mM NaCl, 2.7mM KCl, 8.09mM Na2HPO4, 1.47mM KH2P04, pH7.4)重悬， 负染电镜观察， 分装， 冻存于 _80°C备用。
( 2 ) 将步骤 （1 ) 提纯的 WSSV加入等体积含十二烷基磺酸钠的样品缓冲液， 在沸水中 煮 3-5min后， 加入电泳仪 (Mini -PROTEAN II， Bio- Rad) 上样孔中， 电泳。 取出电泳后的 凝胶放在 CuCl2可逆染色液 （CuCl2 2¾0 5. 11克溶于 100ml双蒸水中） 中染色， 切下 VP28蛋 白条带， 脱色、 洗脱、 透析、 冷冻干燥后， 以 PBS溶解并调整蛋白浓度至 lmg/ml， 分装 50 μ ΐ/管， 冻存于 -80°C。
二、 免疫小鼠
以步骤一提纯的 WSSV-VP28蛋白为抗原， 免疫 Balb/c小鼠， 免疫共分 4次， 前 2次免 疫间隔为 2周， 后 2次免疫间隔为 1周， 前 2次免疫为腹腔注射， 后 2次免疫为尾静脉注 第 1次， 基础免疫， 提纯的 WSSV-VP28蛋白与福氏完全佐剂等体积混匀， 每只注射 100 μ 1；
第 2次， 加强免疫， 提纯的 WSSV-VP28蛋白与福氏不完全佐剂等体积混匀， 每只注射 100 μ 1；
第 3-4次， 加强免疫， 每只注射提纯的 WSSV-VP28蛋白注射 50 μ 1。
三、 细胞融合
( 1 ) 乙醚麻醉免疫小鼠， 无菌取出脾脏和胸腺后， 分别过 100目网筛， 用 RPMI-1640 溶液吹打形成单细胞悬液；
( 2) 分别将脾细胞悬液和胸腺细胞悬液 800g离心 3min， 弃去上清液， 脾细胞沉淀用 RPMI-1640溶液重悬， 胸腺细胞沉淀用含有 1%HAT的 RPMI-1640 (含 10%胎牛血清）选择性细 胞培养液重悬；
( 3) 取 3 X 107个处于对数生长期的 P3-X63-Ag8Ul骨髓瘤细胞， 800g离心 3min， 弃去 上清液后， 用 RPMI-1640溶液重悬；
(4) 将脾细胞悬液与骨髓瘤细胞悬液混合均匀后， 800g离心 3min， 吸去上清液， 轻 弹离心管底，使两种细胞沉淀充分混匀成糊状;用吸管吸取预热到 37°C的聚乙二醇溶液 lml， 缓缓滴加到离心管内； 然后在 37°C水浴中静置 5min;
( 5) 继续滴加已经预热到 37°C的 RPMI-1640溶液 15ml ;
(6) 补加 RPMI-1640溶液至 40ml， 经 800g离心 3min， 弃去上清液；
( 7) 沉淀的细胞以预热到 37°C的 RPMI-1640 (含 10%胎牛血清）细胞培养液 3ml重悬； (8)将细胞悬液加入步骤 (2)制备的胸腺细胞悬液，混合均匀后滴加到 96孔培养板中；
(9)将培养板放入 37°C C02培养箱中培养， 倒置显微镜观察细胞生长情况， 取杂交瘤 细胞培养液检测。
四、 筛选和克隆
( 1 ) 初步筛选
杂交瘤细胞群落长到占 96孔培养板的孔底面积约 1/3时，采用间接免疫荧光法初步筛 选抗 WSSV的阳性杂交瘤细胞株。
① 取感染 WSSV的病虾鳃组织， 生理盐水清洗， 吸干水分后， OCT包埋， 冰冻切片， 切片厚度 5Mffl。 切片贴至干净载玻片上， 自然晾干， 丙酮固定， -20°C保存备用；
② 吸取杂交瘤细胞培养液， 滴加在切片上， 37°C湿盒中孵育 45min;
③ 用含 0. 05%吐温 -20的 PBS (PBST) 洗涤 3次， 每次 5min;
④ 将异硫氰酸荧光素标记的羊抗小鼠 IgG抗体液滴加在切片上， 37°C湿盒中避光孵育 45min; ⑤取出载玻片， 同步骤③洗涤； 封片， 观察。
( 2) 克隆
采用有限稀释法对检测为阳性孔的杂交瘤细胞进行克隆， 步骤如下：
① 乙醚麻醉小鼠， 取胸腺， 制成单细胞悬液；
② 胸腺细胞悬液 800g离心 3min， 弃去上清，沉淀用 10ml RPMI-1640 (含 10%胎牛血清） 细胞培养液重悬；
③ 取阳性孔的杂交瘤细胞约 100个， 加入 10ml胸腺细胞悬液中；
④ 细胞悬液用滴管吹打均匀， 滴加到 96孔培养板中， 每孔 Ιθθμΐ , 平均每个孔约含有 一个杂交瘤细胞；
⑤ 培养板放入 37°C C02培养箱中培养；
⑥ 两周后按照本实施例步骤四 （1 ) 所述方法检测单个克隆孔的杂交瘤细胞培养液；
⑦ 把所得阳性单个克隆孔的杂交瘤细胞按上述方法再克隆一次， 以保证为抗体阳性单 克隆。
( 3) 二次筛选
采用转印免疫印迹法从初步筛选得到的抗 WSSV 的阳性杂交瘤细胞株中筛选抗
WSSV-VP28阳性杂交瘤细胞株：
① 步骤一 （1 ) 中提纯的病毒加入样品缓冲液， 煮沸后进行十二烷基磺酸钠-聚丙烯酰 胺凝胶电泳， 电泳后， 取出凝胶；
② 剪取与电泳凝胶相同大小的硝酸纤维素膜（孔径 0.22μιη)以电转移缓冲液 (25mmol/L Tris-Base, 192mmol/L甘氨酸， 20%甲醇， pH 8.3 )润湿， 剪取比凝胶稍大的滤 纸以电转移缓冲液润湿， 按照滤纸-硝酸纤维素膜 -凝胶 -滤纸的顺序放置， 入盛有电转移缓 冲液的电泳槽内， 将硝酸纤维素膜面向阳极； 200mA电泳 5h;
③ 转移完毕， 取出硝酸纤维素膜； 将硝酸纤维素膜用 PBS洗 10min， 然后放入 3%的 牛血清白蛋白溶液中 37°C封闭 lh;
④ 用 PBST洗涤 3次， 每次 5min;
⑤ 将硝酸纤维素膜置入抗 WSSV抗体阳性杂交瘤细胞培养液中， 37°C孵育 lh， 同④法 洗涤；
⑥ 将硝酸纤维素膜放入碱性磷酸酶标记的羊抗小鼠 Ig抗体（1:4000稀释）中， 37°C孵 育 lh， 同⑥法洗涤；
⑦ 将硝酸纤维素膜放入碱性磷酸酶发色液 （NBT-BCIP发色液） 中发色， 直到颜色清 晰， 去离子水洗涤终止反应。
(4) 三次筛选 采用螯虾体内中和实验筛选具有中和性的抗 WSSV-VP28单克隆抗体：
① 将上述实施例 1中的一 （1 ) 提纯的 WSSV用无菌 PBS 1 : 100稀释， 以 WSSV稀释液 与阳性单克隆杂交瘤细胞培养液等体积混合为实验组； 以 WSSV稀释液与骨髓瘤细胞培养液 等体积混合为阳性对照； 以骨髓瘤细胞培养液为阴性对照； 各组样品室温放置 2h后， 注射 螯虾进行体内中和实验。
② 分别从螯虾的腹面第三腹节皮下注射， 每尾 50 μ 1。
③ 实验组和对照组养殖条件相同，每天换水一次。注射之后每天早晚观察记录死亡数。 结果： 经间接免疫荧光法初步筛选得到的 10株阳性杂交瘤细胞分泌的单抗能与 WSSV 特异性结合，采用有限稀释法对这 10株阳性杂交瘤细胞进行了克隆；通过转印免疫印迹法、 螯虾体内中和实验筛选出了 1 株抗原决定簇位于囊膜蛋白 VP28 上， 具有明显中和性的抗 WSSV-VP28单抗用于抗独特型单抗的研制。 见图 1、 图 2。 图 1 中： M是标准分子量蛋白电 泳后的考马斯亮蓝染色结果； V是提纯 WSSV电泳后的考马斯亮蓝染色结果； 1是将 Abl与 转印后的 WSSV蛋白带反应， Abl识别分子量为 28kDa的蛋白。 图 2中： 阳性对照是将骨髓 瘤细胞培养液与 WSSV稀释液孵育后， 注射入螯虾体内， 15天中螯虾的死亡率变化， 螯虾在 注射后第 1天出现死亡， 第 7天死亡率即达到 100%; 阴性对照是将骨髓瘤细胞培养液注射 入螯虾体内， 15天中螯虾的死亡率变化， 到第 15天时， 螯虾死亡率为 5%; Abl是将 Abl与 WSSV孵育后， 注射入螯虾体内， 15天中螯虾的死亡率变化， 螯虾第 5天出现死亡， 第 15 天死亡率达到 100%。
实施例 2
抗 WSSV-VP28独特型单克隆抗体的制备：
一、 抗原的制备
Balb/c小鼠每只腹腔注射 0. 5ml液体石蜡， 10天后腹腔注射分泌抗 WSSV-VP28单克隆 抗体 (Abl)的杂交瘤细胞， 饲养并密切观察小鼠， 14天左右小鼠腹部膨大后抽取腹水。腹水 经辛酸-硫酸铵法初步纯化后再以 Protein G Agarose (Amersham) 纯化， 调整浓度 2mg/ml， 分装备用。
二、 免疫小鼠
以本实施例一提纯的 Abl作为抗原， 免疫 Balb/c小鼠， 免疫共分 5次进行， 前 2次免 疫间隔为 2周， 后 3次免疫间隔为 1周， 前 2次免疫为腹腔注射， 后 3次免疫为尾静脉注 射。
第 1次， 基础免疫， 提纯的 Abl与福氏完全佐剂等体积混匀， 每只注射 ΙΟΟ μ Ι ;
第 2次， 加强免疫， 提纯的 Abl与福氏不完全佐剂等体积混匀， 每只注射 100 μ ΐ ; 第 3-5次， 加强免疫， 每只注射提纯的 Abl 50 μ 1。 三、 细胞融合
按照实施例 1步骤三所述方法进行细胞融合。
四、 筛选和克隆
( 1 ) 初步筛选
待融合后的杂交瘤细胞群落长到 96孔培养板的孔底面积约 1/3时开始检测，采用间接 酶联免疫吸附法初步筛选抗 WSSV-VP28抗独特型单克隆抗体阳性杂交瘤细胞。
① 以纯化的 WSSV-VP28蛋白免疫新西兰白兔， 制备兔抗 WSSV-VP28抗体， 抗血清经辛 酸硫酸铵法纯化， 用碳酸盐包被液 （pH 9. 6) 稀释至蛋白浓度 0. 02mg/ml， 以每孔 ΙΟθμΙ加 入 96孔酶标板， 4°C包被过夜；
② 吸出包被液， 用 PBST洗涤 3次， 每次 5min;
③ 每孔加入 200μ1 3%的牛血清白蛋白， 37°C封闭 lh;
④ 同步骤②洗涤；
⑤ 每孔加入杂交瘤细胞培养液 Ιθθμΐ ,以骨髓瘤细胞培养液为阴性对照， 37°C孵育 lh;
⑥ 同步骤②洗涤；
⑦ 每孔加入碱性磷酸酶标记的羊抗小鼠 Ig ( 1 : 4000稀释） Ιθθμΐ , 37°C孵育 lh;
⑧ 同步骤②洗涤后， 每孔加入 Ιθθμΐ 4-硝基酚磷酸盐 （ρΝΡΡ) 发色液， 暗处反应 5— 30min， 每孔加入 50μ1 2Μ的 NaOH终止发色， 稳定 3_5min， 以酶标仪测各孔 405nm波长处 的 0D值。 计算各实验孔与阴性对照 0D值之比 （P/N)， 当 P/N 2. 1时为阳性。
( 2) 克隆： 按照实施例 1四 （2 ) 中步骤①-⑤对检测出为阳性孔的杂交瘤细胞进行克 隆。 两周后按照本实施例四 （1 ) 步骤①-⑧检测单个克隆孔的杂交瘤细胞培养液， 把所得 阳性单个克隆孔的杂交瘤细胞再克隆一次， 以保证为抗体阳性单克隆。
( 3) 二次筛选
初步筛选得到的阳性杂交瘤细胞共 6株 （分别命名为单抗 A、 B、 C、 D、 E、 F) 分别注 入小鼠腹腔生产腹水， 采用竞争酶联免疫吸附法进行二次筛选：
① 实施例 1一（1 )中提纯的 WSSV用碳酸盐包被液 1 : 40稀释， WSSV稀释液以每孔 ΙΟθμΙ 加入 96孔酶标板中， 4°C包被过夜；
② 吸出包被液， 用 PBST洗涤 3次， 每次 5min;
③ 每孔加入 200μ1 3%的牛血清白蛋白， 37°C封闭 lh;
④ 同步骤②洗涤；
⑤调整兔抗 WSSV-VP28抗体浓度为 40 μ g/ml， 单抗 A、 B、 C、 D、 E、 F分别以 PBS梯度 稀释 10、 100、 1000倍， 抗体稀释液分别与兔抗 WSSV-VP28抗体等体积混合为实验组， 4°C 放置过夜后， 每孔加入 Ιθθμΐ混合液， 37°C孵育 lh， 以 PBS代替单抗为无竞争对照组； ⑥ 同步骤②洗涤；
⑦ 每孔加入 Ιθθμΐ碱性磷酸酶标记的羊抗兔 Ig ( 1 : 4000稀释）， 37°C孵育 lh;
⑧ 同步骤②洗涤；每孔加入 Ιθθμΐ 4-硝基酚磷酸盐 (ρΝΡΡ)发色液，暗处反应 5— 30min， 每孔加入 50μ1 2Μ的 NaOH终止发色， 稳定 3— 5min， 以酶标仪测各孔 405nm波长处的 0D值。 按照抑制率 = ( 1 - OD ffl/ OD无鮮應 X 100%的公式计算抑制率。
结果： 筛选出的单抗 A能与 WSSV竞争结合兔抗 WSSV-VP28抗体， 随着抗体稀释倍数的 增加， 抑制率降低， 见图 3。 单抗 A是由保藏号为： CCTCC一 C200938的杂交瘤细胞分泌的， 该单抗为抗 WSSV-VP28独特型单克隆抗体。
五、 冻存
取生长旺盛， 形态良好的单抗 A细胞， 制成细胞悬液， 200g离心 5min， 去上清， 加冻存 液 （9份 RPMI-1640培养基十 1份二甲亚砜）， 使最终细胞密度为 5 X 106个 /ml， 将 1ml细胞 悬液装于 2ml冻存管中， 拧紧螺盖， 放于 -80°C超低温冰箱内过夜（8-12h)后， 再浸入液氮 内长期保存。 本发明获得生长旺盛， 形态良好的杂交瘤细胞， 定名为： 杂交瘤细胞 AMVP, 保藏号为： CCTCC一 C200938, 保藏日期为： 2009年 5月 11 日。
实施例 3
本发明抗 WSSV-VP28独特型单克隆抗体的竞争酶联免疫吸附法鉴定：
① 实施例 1步骤一 （1 ) 提纯的 WSSV以碳酸盐包被液 1 : 40稀释， 以每孔 Ιθθμΐ加入 96孔酶标板中， 4°C包被过夜；
② 吸出包被液， 用 PBST洗涤 3次， 每次 5min;
③ 每孔加入 200μ1 3%的牛血清白蛋白， 37°C封闭 lh;
④ 同步骤②洗涤；
⑤ 以辛酸-硫酸铵法纯化的本发明单抗为抗原， 免疫 Balb/c小鼠获得抗血清 （Ab3)， 本发明单抗和 Ab3分别以 PBS梯度稀释 10、 20、 40、 80、 160、 320、 640、 1 280、 2 560、 5120倍，本发明单抗和 Ab3的各梯度稀释液再分别与浓度为 40 μ g/ml的兔抗 WSSV-VP28抗 体等体积混合为实验组， 4°C过夜后， 每孔加入 Ιθθμΐ混合液， 37°C孵育 lh， 以 PBS代替本 发明单抗和 Ab3为无竞争对照；
⑥其余步骤及结果判定同实施例 2中的四 （3 ) 步骤⑥ -⑧。
结果： 本发明单抗及 Ab3均能竞争抑制兔抗 WSSV-VP28抗体与 WSSV的结合， 随着稀释 倍数的增加， 抑制率降低， 见图 4。
实施例 4
抗 WSSV-VP28单克隆抗体 （Abl ) 与抗抗 WSSV-VP28独特型抗体 （Ab3 ) 的间接免疫荧 光法鉴定： 将 Abl与 Ab3分别滴加于实施例 1中的四 （1 )步骤①制备的切片上， 37°C湿盒中孵育 45min, 其余步骤同实施例 1中的四 （1 ) 步骤③ -⑤。
结果： Abl和 Ab3都能与 WSSV发生特异性结合， 再与荧光素标记的羊抗小鼠 IgG抗体 结合， 感染 WSSV的病虾鳃丝上可见黄绿色荧光， 见图 5a、 5b。 表明 Ab3能与 WSSV结合， 具有 Abl特性。 图 5 中： a是感染 WSSV的病虾鳃与 Abl孵育后， 再与荧光素标记的羊抗小 鼠 IgG抗体孵育， 可观察到黄绿色荧光。 b是感染 WSSV病虾的鳃与 Ab3孵育后， 再与荧光 素标记的羊抗小鼠 IgG抗体孵育， 可观察到黄绿色荧光。 图中标尺为 50Mffl。
实施例 5
抗 WSSV-VP28单克隆抗体 （Abl ) 与抗抗 WSSV-VP28独特型抗体 （Ab3 ) 的金标记免疫 电镜法鉴定：
( 1 ) 吸取实施例 1 中的一 （1 ) 提纯的 WSSV1(^1， 滴在覆有支持膜的铜网上， 吸附 10-15min， 多余的液体用滤纸吸干。
( 2 ) 吸取 Abl和 Ab3 各 Ιθμΐ分别滴在铜网上， 室温结合 20min;
( 3 ) 用 PBS冲洗 5次；
( 4) 吸取胶体金标记的羊抗小鼠 IgG抗体 Ιθμΐ滴在铜网上， 室温结合 30min;
( 5 ) 同步骤 （3 ) 冲洗， 再经双蒸水冲洗 2次， 用滤纸吸干；
( 6 ) 用 2%磷钨酸 （pH 6. 5 ) 负染约 30秒， 用滤纸吸干， 电镜观察。
结果： 在 WSSV粒子上结合有胶体金粒子， 此结果直接证实 Abl和 Ab3的抗原决定簇位 于 WSSV的囊膜上， 见图 6b、 6c。 图 6中： a是提纯 WSSV的电镜照片， 病毒粒子纯度高， 完整， 具囊膜。 b中可见胶体金颗粒结合在 WSSV囊膜上， 此结果证明 Abl的抗原决定簇位 于囊膜上。 c中可见胶体金颗粒结合在 WSSV囊膜上，此结果证明 Ab3的抗原决定簇位于 WSSV 囊膜上。 图中标尺为 100nm。
实施例 6
按照实施例 1中的四 （四） 所述方法采用螯虾体内中和实验证实抗抗 WSSV-VP28独特 型抗体 （Ab3 ) 的中和性。 将实施例 1中的一 （1 ) 提纯的 WSSV用无菌 PBS 1 : 100稀释； 以 WSSV稀释液与 Ab3等体积混合为实验组； 以 WSSV与正常小鼠血清等体积混合为阳性对照； 以正常小鼠血清为阴性对照， 各组样品室温放置 2h后， 注射螯虾进行体内中和实验。 其余 步骤同实施例 1中的四 （4) 步骤② -③。
本发明所采用的螯虾体内中和实验结果： 阴性对照是将正常小鼠血清注射入螯虾体内， 15天中螯虾的死亡率变化，螯虾 15天中无死亡；阳性对照是将与正常小鼠血清孵育的 WSSV 注射入螯虾体内， 15天中螯虾的死亡率变化， 螯虾在注射 WSSV后第 1天开始死亡， 第 7天 死亡率达 100%; Ab3是将与 Ab3孵育的 WSSV注射入螯虾体内， 15天中螯虾的死亡率变化， 螯虾在注射后第 5天开始死亡， 直到第 15天， 仍有近 20%的螯虾存活， 见图 7。
本领域的普通技术人员都会理解， 在本发明的保护范围内， 对于上述实施例进行修改， 添加和替换都是可能的， 都没有超出本发明的保护范围。

Claims

权 利 要 求 书
1、 一种抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体， 其特征在于： 所述的单克 隆抗体是由保藏号为： CCTCC一 C200938的杂交瘤细胞 AMVP分泌的。
2、 一种如权利要求 1所述抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体的制备方法， 其特征在于包括以下步骤：
( 1 ) 纯化经体内、 体外中和实验验证具有中和性的抗 WSSV-VP28单克隆抗体为抗原， 免疫 Balb/c小鼠， 取脾脏获得脾细胞；
(2)将小鼠骨髓瘤细胞与脾细胞融合， 得到融合的杂交瘤细胞；
(3)采用免疫学的筛选方法，筛选得到分泌抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆 抗体的杂交瘤细胞；
(4)克隆得到单克隆细胞株，用常规方法培养， 收集细胞培养液或注入小鼠腹腔制备腹水， 以硫酸铵沉淀和亲和层析方法纯化得到抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体。
3、如权利要求 2所述抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体的制备方法，其特 征在于： 所述的免疫学的筛选方法为间接酶联免疫吸附法和竞争酶联免疫吸附法。
4、如权利要求 2所述抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体的制备方法，其特 征在于以纯化的抗 WSSV-VP28单克隆抗体 (2mg/ml )为抗原按如下方式免疫： 共分 5次进行， 第 1次， 基础免疫， 抗原与福氏完全佐剂等体积混匀， 腹腔注射， 每只注射 100 μ ΐ; 第 2次， 加强 免疫， 抗原与福氏不完全佐剂等体积混匀， 腹腔注射， 每只注射 100 μ ΐ; 后 3次为尾静脉注射， 不用佐剂， 每只注射 50 μ ΐ ; 前 2次免疫间隔为 2周， 后 3次免疫间隔为 1周； 取免疫后的小鼠 脾脏以获得脾细胞。
5、如权利要求 3所述抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体的制备方法，其特 征在于所述的间接酶联免疫吸附法包括以下步骤： 以纯化的 WSSV-VP28蛋白免疫新西兰白兔制备 的兔抗 WSSV-VP28抗体稀释至 0. 02mg/ml， 以每孔 ΙΟθμΙ 加入 96孔酶标板中， 4°C包被过夜； 吸 出包被液， PBST洗漆 3次， 每次 5min; 每孔加入 200μ1 3%的牛血清白蛋白， 37°C封闭 lh; 洗 涤 3次后， 每孔加入杂交瘤细胞培养液 Ιθθμΐ , 以骨髓瘤细胞培养液为阴性对照， 37°C孵育 lh; 洗涤 3次后， 每孔加入碱性磷酸酶标记的羊抗小鼠 Ig ( 1 : 4000稀释） Ιθθμΐ, 37°C孵育 lh; 每 孔加入 4-硝基酚磷酸盐 （pNPP) 发色液 Ιθθμΐ, 暗处反应 5—30min， 每孔加入 50μ1 2Μ的 NaOH 终止发色， 稳定 3— 5min， 以酶标仪测各孔 405nm波长处的 0D值， 当 P/N 2. 1时为阳性。
6、如权利要求 3所述抗对虾白斑症病毒囊膜蛋白 VP28独特型单克隆抗体的制备方法， 其特 征在于所述的竞争酶联免疫吸附法包括以下步骤：间接酶联免疫吸附法筛选出的阳性杂交瘤细胞 注入经液体石蜡处理的小鼠腹腔内制备腹水， 14天左右小鼠腹部膨大后收集腹水； 提纯的 WSSV 用碳酸盐包被液稀释后， 以每孔 100 μ 1加入 96孔酶标板， 4°C包被过夜； 吸出包被液， 用 PBST 洗漆 3次， 每次 5min; 以 3%的牛血清白蛋白， 37°C封闭 lh; 洗漆 3次； 调整兔抗 WSSV-VP28抗 体浓度为 40 y g/ml， 单抗 A、 B、 C、 D、 E、 F以 PBS梯度稀释 10、 100、 1000倍， 抗体稀释液分别 与兔抗 WSSV-VP28抗体等体积混合为实验组， 4°C放置过夜后， 每孔加入 Ιθθμΐ混合液， 37°C孵育 lh, 以 PBS代替单抗为无竞争对照； 洗涤 3次后， 每孔加入碱性磷酸酶标记的羊抗兔 Ig ( 1 : 4000 稀释） Ιθθμΐ, 37°C孵育 lh; 洗涤 3次后， 每孔加入 4_硝基酚磷酸盐 （pNPP) 发色液 100μ1， 暗 处反应 5—30min， 每孔加入 50μ1 2Μ的 NaOH终止发色， 稳定 3—5min， 以酶标仪测各孔 405應波 长处的 0D值； 按照抑制率 = ( l_OD ffl/ OD无鮮对隨） X 100%计算抑制率， 筛选竞争抑制效果明显 的单抗。
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