WO2019237442A1 - 抗人b7-h4单克隆抗体、鉴定方法、应用和试剂盒 - Google Patents

抗人b7-h4单克隆抗体、鉴定方法、应用和试剂盒 Download PDF

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WO2019237442A1
WO2019237442A1 PCT/CN2018/094692 CN2018094692W WO2019237442A1 WO 2019237442 A1 WO2019237442 A1 WO 2019237442A1 CN 2018094692 W CN2018094692 W CN 2018094692W WO 2019237442 A1 WO2019237442 A1 WO 2019237442A1
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chain variable
variable region
monoclonal antibody
human
light chain
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张学光
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苏州旭光科星生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

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  • the invention relates to an antibody immunohistochemical detection application, and the verification of the antibody heavy chain variable region and light chain variable region sequences.
  • the invention belongs to the field of biotechnology, and particularly relates to an anti-human B7-H4 monoclonal antibody, an identification method, and applications. And kit.
  • B7-H4 molecules are abnormally expressed in various tumor tissues, such as ovarian cancer, breast cancer, lung cancer, kidney cancer, colorectal cancer, and prostate cancer.
  • Clinical retrospective case analysis found that there was a correlation between B7-H4 expression and clinicopathological features. Survival analysis revealed that cases with high B7-H4 expression had poor prognosis, high mortality, and high levels in peripheral blood of tumor patients. Levels of soluble B7-H4 (sB7-H4).
  • SB7-H4 soluble B7-H4
  • B7-H4 is associated with transplant immune rejection and autoimmune pathology. Transplants transfected with the B7-H4 gene can inhibit the activity of host T cells, thereby extending the survival time of the transplant.
  • B7-H4 is expressed at mRNA and protein levels and is negatively correlated with patient prognosis.
  • B7-H4 is abnormally expressed in tumor tissue or peripheral blood of tumor patients, and is closely related to age, pathological type, tumor biological effect and patient survival rate. Therefore, the development of mouse anti-human B7-H4 monoclonal antibody can be further researched.
  • This molecule lays a material foundation for the role of tumors, and at the same time provides an effective research means for exploring B7-H4 signaling pathways and mechanisms. Functional anti-human B7-H4 monoclonal antibodies are more likely to become potential targets for tumor biotherapy point.
  • An object of the present invention is to provide an anti-human B7-H4 monoclonal antibody
  • Another object of the present invention is to provide a method for identifying heavy chain and light chain variable region sequences of anti-human B7-H4 antibodies.
  • the present invention extracts heavy chain variable regions (mVH) and light chain variable regions (mVL) from hybridoma cells that secrete anti-human B7-H4 monoclonal antibodies, and retains candidate heavy and light chain variable region sequences according to sequencing results.
  • the heavy chain and light chain variable region sequences matched with the expression vector were amplified by PCR, and the PCR product was ligated with a double-digested pre-treated linear expression vector. The ligated product transformed the competent bacteria DH5a.
  • the eukaryotic expression cell line 293 was co-transfected with the expression vector linked to the heavy chain and light chain variable region genes of the monoclonal antibody of interest, and the supernatant obtained from the culture contained the antibody of interest, which proved that the obtained heavy chain and light chain variable region sequences were correct;
  • the third object of the present invention is to provide an anti-human B7-H4 monoclonal antibody for use in immunohistochemical detection of tissue specimens. It is found that the antibody B7-H4 is used for histochemical staining of liver cancer tissue specimen tissues. Significant staining; further detection of immunohistochemical pathological specimens, the results showed that B7-H4 was highly expressed in the cytoplasm and nucleus of mesenchymal cells and the cell membrane of intestinal cancer patients.
  • the fourth object of the present invention is to provide a kit prepared by using the above-mentioned anti-human B7-H4 monoclonal antibody.
  • an anti-human B7-H4 monoclonal antibody includes a heavy chain and a light chain, characterized in that the amino acid sequence of the heavy chain variable region (mVH) is the same as SEQ ID NO.1, details as follows:
  • amino acid sequence of the light chain variable region (mVL) is the same as SEQ ID NO. 2, as follows:
  • the heavy chain variable region has three hypervariable regions: GYSITSDFA, ISYSGDT, ARDYGSGAWYFDV.
  • the light chain variable region has three hypervariable regions: QSLLNSRTRKNY, WAS, and KQSYNLWT.
  • a method for identifying heavy and light chain variable region sequences of an anti-human B7-H4 antibody is characterized in that it includes the following steps:
  • Extracting the cDNA of the hybridoma cell extract RNA from the hybridoma cell line, and use RT-PCR technology to reverse transcribe the obtained RNA into cDNA;
  • the specifically designed upstream and downstream primers were used to clone the heavy chain variable region (mVH) and light chain variable region (mVL) of the hybridoma cell; the sequences of the specifically designed upstream and downstream primers were nucleotides SEQ ID ID NO .3 and nucleotide sequence 4;
  • the heavy chain variable region (mVH) and light chain variable region (mVL) are respectively linked to a cloning vector (pJET, cloning vector), and the ligation product transforms the competent bacteria DH5a, because the pJET vector carries ampicillin (Amp +) Resistance gene, the transformed bacteria liquid can be coated on Amp-resistant LB solid medium, and cultured at 37 ° overnight;
  • Retain candidate heavy chain variable region (mVH) and light chain variable region (mVL) sequences based on sequencing results, and PCR-amplify the heavy chain variable region (mVH) and light chain
  • the variable region (mVL) sequence is used to link the PCR product with a double-digested pre-treated linear expression vector.
  • the ligation product transforms the competent bacteria DH5a. Since the expression vector contains a kanamycin (Kana +) resistance gene, the transformed bacteria can be transformed.
  • the solution was spread on Kana-resistant LB solid medium, and cultured at 37 ° overnight;
  • the eukaryotic expression cell line 293 is a suspension culture, and SFM4Transfx-293 without L-glutamine (liquid) in serum-free medium is passaged and amplified, and transfected.
  • FreeStyle TM 293 Expression Medium was replaced by serum-free medium; the supernatant was harvested after 7 days of continuous culture, centrifuged at 4000g for 30min, the impurities such as cells in the supernatant were removed, and the bacteria were filtered through a 0.45um filter.
  • the anti-human B7-H4 monoclonal antibody is used to prepare a kit for immunohistochemical detection of tumor tissue.
  • kits are characterized in that the kit contains the above-mentioned anti-human B7-H4 monoclonal antibody.
  • the anti-human B7-H4 monoclonal antibody provided by the present invention can be used for the immunohistochemical detection of tumor tissues, improve the accuracy of immunohistochemical detection of tumor specimens, and help to diagnose malignant tumors, determine the primary site and pathological typing To improve the diagnostic accuracy of tumors, especially poorly differentiated or undifferentiated tumors.
  • Figure 1 is a schematic diagram of Western blot analysis of B7-H4 protein by anti-human B7-H4 monoclonal antibody 4B2.
  • the results in the figure show that monoclonal antibody 4B2 can specifically recognize and bind to the B7-H4 protein in cell lysate with a clear band , The negative control Isotype and CHO / mock groups did not bind bands.
  • IgG Isotype negative antibody isotype control
  • 4B2 anti-human B7-H4 monoclonal antibody of the present invention
  • CHO / mock cell lysate CHO / B7-H4 cell lysate.
  • Figure 2 is a schematic diagram of the karyotype analysis ( ⁇ 1000 times) of the obtained hybridomas using nuclear chromosome technology.
  • the results of the karyotype analysis of the hybridoma cells in the figure show that the number of chromosomes of the hybridoma cell lines is more than 80, which exceeds that of mouse B cells. And SP2 / 0 cell chromosome number, indicating that it is a fused cell.
  • FIG. 3 is a schematic diagram of anti-human B7-H4 antibody used for immunohistochemical detection of liver cancer tissues.
  • 4B2 immunohistochemical detection of liver cancer tissue specimens CST is a positive control; the results show that B7-H4 is highly expressed in liver cancer tissues, and CST and 4B2 antibodies All tissues are stained.
  • Figure 4 is a schematic diagram of specimens of anti-human B7-H4 antibodies used for immunohistochemical detection of clinical intestinal cancer patients: the figure shows that the anti-human B7-H4 molecules are basically not expressed in the benign lesion stage, and are scattered in the colorectal inflammation tissue, and their expression The adenoma stage is gradually upregulated and is highly expressed in colorectal cancer tissues.
  • the sources of biological materials used in the examples are as follows:
  • mice were immunized with fusion protein or transgenic cells, four times at 21-day intervals, and the orbital blood titer of the mice was measured after the fourth immunization. The titer was determined to strengthen the immunity.
  • the spleen of the mouse was removed aseptically, placed in a 200 mesh stainless steel sieve, and ground to obtain a single cell suspension. Wash twice with 1640 basal medium (1400 rpm, 5 min) for later use. 1640 medium with 15% FBS was used to adjust the cell concentration to 2 ⁇ 10 5 / ml, and the solution was added dropwise to a 96-well culture plate, 100 ⁇ L per well, and cultured in a 37 ° C., 5% CO 2 incubator.
  • Equipment mouse cup; simple dissection table; hybridoma package; insulated water bath cup; thermometer; 96-well culture plate.
  • Reagent 75% ethanol solution; preheat 1640 basal medium at 37 ° C; preheat 1ml PEG at 37 ° C; preheat 16ml basal medium 14ml at 37 ° C (PEG stop solution); preheat 1640 selection medium (containing 15 % FBS, and the amount of HAT added was calculated such that the final concentration of HAT in the medium in the final 96-well culture plate was 1%).
  • mice 1. Immunize the mice, rinse them with running water, and place them in 75% ethanol solution for 2 min.
  • the spleen of the mouse was removed aseptically, placed in a 200 mesh stainless steel sieve, and ground to obtain a single cell suspension. Wash twice with preheated 1640 basal medium (1400 rpm, 5 min) for use.
  • RNA is extracted from this hybridoma cell line, and the obtained RNA is reverse transcribed into cDNA using RT-PCR technology; the hybridoma cells are cloned by PCR using specific designed upstream and downstream primers.
  • mVH and mVL were respectively linked to the cloning vector (pJET, cloning vector), and the ligation product transformed the competent bacteria DH5a. Since the pJET vector contains the ampicillin (Amp +) resistance gene, the transformed bacterial liquid can be coated on the Amp-resistant LB solid Incubate overnight at 37 ° on the medium;
  • the candidate heavy and light chain variable region sequences are retained according to the sequencing results, and the heavy and light chain variable region sequences that match the expression vector are amplified again by PCR, and the PCR product is ligated to the pre-treated linear expression vector with double enzyme digestion.
  • the ligation product transforms the competent bacteria DH5a. Because the expression vector carries the kanamycin (Kana +) resistance gene, the transformed bacterial solution can be coated on Kana-resistant LB solid medium and cultured at 37 ° overnight;
  • Eukaryotic expression cell line 293 was co-transfected with the expression vector linked to the heavy chain and light chain variable region genes of the monoclonal antibody of interest.
  • 293 cells were cultured in suspension and SFM4Transfx-293 without L-glutamine (liquid) in serum-free medium Amplification, for transfection FreeStyle TM 293 Expression Medium serum-free medium replacement. After 7 days of continuous culture, the supernatant was harvested, centrifuged at 4000g for 30min, and impurities such as cells in the supernatant were removed, and sterilized by filtering with a 0.45um filter;
  • the harvested supernatant contained the antibody of interest, and Western blot detection showed that the monoclonal antibody was well bound to the transgenic cell CHO / B7-H4.
  • Anti-human B7-H4 antibody heavy chain variable region obtained by the above method:
  • GYSITSDFA Three highly variable regions: GYSITSDFA, ISYSGDT, ARDYGSGAWYFDV.
  • Antibody light chain variable region

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Abstract

本发明公开了一种抗人B7-H4单克隆抗体、抗人B7-H4单克隆抗体、鉴定方法、应用和试剂盒，本发明的抗人B7-H4单克隆抗体以及这株单克隆抗体可用于免疫组化标本检测及肿瘤组织B7-H4表达分析，并从分泌抗人B7-H4单克隆抗体的杂交瘤细胞中提取重链可变区(mVH)和轻链可变区(mVL)，测序验证B7-H4重轻链可变序列。在此基础上实施真核细胞株表达抗体，所获抗体经验证具有良好的结合能力，采用免疫组织化学检测对肿瘤标本特异性染色，并比较自行研制的B7-H4单抗与商品化B7-H4抗体对同一组织标本染色的效果和特异性。

Description

抗人B7-H4单克隆抗体、鉴定方法、应用和试剂盒 技术领域
本发明涉及一种抗体免疫组化的检测应用，以及抗体重链可变区和轻链可变区序列的验证，属于生物技术领域，特别涉及抗人B7-H4单克隆抗体、鉴定方法、应用和试剂盒。
背景技术
近年来，越来越多的研究发现，B7-H4分子异常表达在各种肿瘤组织，如卵巢癌、乳腺癌、肺癌、肾癌、结直肠癌、前列腺癌等。临床回顾性病例分析发现，B7-H4的表达与临床病理学特征存在相关性，通过生存分析发现高表达B7-H4的病例预后差，死亡率高，并且在肿瘤患者外周血中也发现了高水平的可溶性B7-H4(sB7-H4)。此外，研究还发现，B7-H4与移植免疫排斥和自身免疫病理相关，转染了B7-H4基因的移植物可以抑制宿主T细胞的活性，从而延长移植物的生存时间。这些研究结果都提示，B7-H4是个重要的负性共刺激分子，具有重要的研究和运用价值。
人类很多肿瘤中，B7-H4在mRNA和蛋白水平上均有表达并且与病人的预后呈负相关。B7-H4异常表达于肿瘤组织或肿瘤患者的外周血中，并和年龄、病理类型、肿瘤生物学效应和患者生存率密切相关，因此，研制鼠抗人B7-H4单克隆抗体可以为进一步研究该分子在肿瘤中的作用奠定物质基础，同时也为探明B7-H4信号传导通路及机制提供有效的研究手段，有功能的抗人B7-H4单克隆抗体更有望成为肿瘤生物治疗的潜在靶点。
发明内容
本发明的目的之一是提供一种抗人B7-H4单克隆抗体；
本发明的目的之二是提供一种抗人B7-H4抗体重链和轻链可变区序列的鉴定方法。本发明从分泌抗人B7-H4单克隆抗体的杂交瘤细胞中提 取重链可变区(mVH)和轻链可变区(mVL)，根据测序结果保留候选的重轻链可变区序列，PCR扩增出与表达载体匹配的重链、轻链可变区序列，将PCR产物与双酶切预先处理的线性表达载体连接，连接产物转化感受态细菌DH5a。连接有目的单抗重链和轻链可变区基因的表达载体共转染真核表达细胞株293，培养收获的上清含有目的抗体，证明得到的重链和轻链可变区序列正确；
本发明的目的之三是提供一种抗人B7-H4单克隆抗体用于免疫组化检测组织标本，发现用该抗体B7-H4对肝癌组织标本组织进行组化染色，胞膜和胞浆均有明显着色；进一步用于免疫组化的病理标本检测，结果显示B7-H4在肠癌病人间质细胞的胞浆和细胞核及肠癌细胞的胞膜呈高表达。
本发明的目的之四是提供一种利用上述抗人B7-H4单克隆抗体制备的试剂盒。
为了实现上述目的，本发明的技术方案如下：
作为本发明的第一方面一种抗人B7-H4单克隆抗体，包括重链和轻链，其特征在于，所述重链可变区(mVH)的氨基酸序列与SEQ ID NO.1相同，具体如下：
Figure PCTCN2018094692-appb-000001
YYCARDYGSGAWYFDVWGAGTTVTVSS；
所述轻链可变区(mVL)的氨基酸序列与SEQ ID NO.2相同，具体如下：
Figure PCTCN2018094692-appb-000002
CKQSYNLWTFGGGTKLEIK。
在本发明的一个优选实施例中，所述重链可变区(mVH)具有三个高变区为：GYSITSDFA、ISYSGDT、ARDYGSGAWYFDV。
在本发明的一个优选实施例中，所述轻链可变区(mVL)具有三个高 变区为：QSLLNSRTRKNY、WAS、KQSYNLWT。
作为本发明的第二方面的一种抗人B7-H4抗体重链和轻链可变区序列的鉴定方法，其特征在于，包括以下步骤：
(1)B7-H4单克隆抗体的杂交瘤的制备；
(1.1)免疫小鼠；
(1.2)细胞培养；
(1.3)融合与筛选；
(2)B7-H4鼠/人嵌合抗体的制备；
(2.1)提取杂交瘤细胞的cDNA：从该杂交瘤细胞株中提取RNA，并使用RT-PCR技术，将获得的RNA反转录为cDNA；
利用特定设计的上下游引物PCR克隆该杂交瘤细胞的重链可变区(mVH)和轻链可变区(mVL)；所述特定设计的上下游引物的序列分别为核苷酸SEQ ID NO.3和核苷酸序列4；
(2.2)所述重链可变区(mVH)和轻链可变区(mVL)分别与克隆载体(pJET cloning vector)连接，连接产物转化感受态细菌DH5a，由于pJET载体带有氨苄(Amp+)抗性基因，可将转化菌液涂在Amp抗性的LB固体培养基上，37°过夜培养；
(2.3)待涂板细菌长出分散菌落，选择边缘清晰、生长良好的菌落，进一步测序鉴定；
(2.4)根据测序结果保留候选的重链可变区(mVH)和轻链可变区(mVL)序列，再次PCR扩增出与表达载体匹配的重链可变区(mVH)和轻链可变区(mVL)序列，将PCR产物与双酶切预先处理的线性表达载体连接，连接产物转化感受态细菌DH5a，由于表达载体带有卡那霉素(Kana+)抗性基因，可将转化菌液涂在Kana抗性的LB固体培养基上，37°过夜培养；
(2.5)测序方法参照(2.4)对比两次测序结果，挑选正确序列的转化菌，扩大培养后进行质粒抽提；
(2.6)连接有目的单抗重链可变区(mVH)和轻链可变区(mVL)基因的表达载体共转染真核表达细胞株293；
(2.7)收获的上清含有目的抗体，流式检测表达单抗与M435结合良好，阳性率达95％以上，表明已获得正确的抗体重链和轻链可变区序列。
在本发明的一个优选实施例中，其中，步骤(2.6)中，所述真核表达细胞株293为悬浮培养，SFM4Transfx-293 without L-glutamine(liquid)无血清培养基传代扩增，转染时用
Figure PCTCN2018094692-appb-000003
FreeStyle TM293 Expression Medium无血清培养基替换；通过7天连续培养后收获上清，4000g离心30min，去除上清中细胞等杂质，并用0.45um滤器过滤除菌。
作为本发明的第三方面一种抗人B7-H4单克隆抗体的应用，其特征在于，所述抗人B7-H4单克隆抗体用于制备用于肿瘤组织的免疫组化检测的试剂盒。
作为本发明的第四方面的一种试剂盒，其特征在于，所述试剂盒包含上述抗人B7-H4单克隆抗体。
本发明的有益效果：
本发明提供的抗人B7-H4单克隆抗体可以用于肿瘤组织的免疫组化检测，提高肿瘤标本免疫组化检测的精确度，有助于恶性肿瘤的诊断、确定原发部位和病理分型，提高肿瘤尤其是低分化或未分化肿瘤的诊断准确率。
附图说明
图1为Western blot分析抗人B7-H4单抗4B2对B7-H4蛋白的识别示意图，图中结果显示，单抗4B2能特异性识别结合细胞裂解液中的B7-H4蛋白，目的条带清晰，阴性对照的Isotype和CHO/mock组没有结合条带。图中：IgG Isotype:同型抗体阴性对照；4B2:本发明抗人B7-H4单克隆抗体；CHO/mock细胞裂解液，CHO/B7-H4细胞裂解液。
图2为采用细胞核染色体技术对获得的杂交瘤进行核型分析(×1000倍)示意图，图中杂交瘤细胞核型分析结果显示，杂交瘤细胞株的染色体数目在80条以上，超过小鼠B细胞和SP2/0细胞的染色体数，表明其为融合细胞。
图3为抗人B7-H4抗体用于免疫组化检测肝癌组织示意图，图中4B2免疫组化检测肝癌组织标本，CST为阳性对照；结果显示B7-H4在肝癌组织高表达，CST和4B2抗体对组织均有着色。
图4为抗人B7-H4抗体用于免疫组化检测临床肠癌病人标本示意图：图中显示抗人B7-H4分子在良性病变阶段基本不表达，在结直肠炎症组织散在性表达，其表达腺瘤阶段逐步上调，在结直肠癌组织中高表达。
具体实施方式
以下结合具体实施例，对本发明作进步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例中如无特殊说明，均为本领域常规实验技术。
实施例中所用生物材料来源如下：
实施例1 产生抗人B7-H4单克隆抗体的杂交瘤的获得
(一)免疫小鼠
用融合蛋白或转基因细胞免疫小鼠，免疫四次，每次间隔21天，第四次免疫7-10后测小鼠眼眶血效价，效价测定好则加强免疫。
(二)细胞培养
1.于融合前1d，取6～7周龄BALB/c小鼠1只，置于75％乙醇溶液中2min。
2.无菌取出小鼠脾脏，置于200目的不锈钢筛网中，研磨获得单个细胞悬液。用1640基础培养基洗涤两遍(1400rpm，5min)备用。用15％FBS的1640培养基，调整细胞浓度为2×10 5/ml，滴加入96孔培养板中，每孔100μL，37℃、5％CO 2培养箱中培养。
3.过夜培养，第二天在低倍显微镜下观察。并铺亚克隆细胞150ul。
(三)融合与筛选
准备：
1.器材：泡老鼠杯；简易解剖台；杂交瘤包；保温水浴杯；温度计； 96孔培养板。
2.试剂：75％乙醇溶液；37℃预热1640基础培养基；37℃预热PEG1ml；37℃预热1640基础培养基14ml(PEG终止液)；37℃预热1640选择培养基(含15％FBS，并计算加入HAT的量，使得最终96孔培养板中培养基的HAT终浓度为1％)。
步骤：
1.将免疫小鼠，用流水冲洗，置于75％乙醇溶液中2min。
2.无菌取出小鼠脾脏，置于200目的不锈钢筛网中，研磨获得单个细胞悬液。用预热1640基础培养基洗涤两遍(1400rpm，5min)备用。
3.收集生长良好、处于对数生长期的SP2/0细胞,用预热1640基础培养基洗涤两遍(1400rpm，5min)备用。
4.将SP2/0细胞或Ag8细胞与脾脏细胞混合于50ml透明塑料离心管中，一般脾细胞:骨髓瘤细胞比例为5:1，预热1640基础培养基洗涤一遍(1400rpm，5min)，弃尽上清(以避免对PEG产生不必要的稀释)并用掌心搓管底(或手指轻轻弹击管底)，使两种细胞充分混匀成悬浮细胞状。
5.将离心管置于37℃保温水浴杯中预热，吸取1ml经37℃预温的50％的PEG溶液，在1min内匀速加完，且边加边轻轻震荡离心管，加完后在37℃水浴中轻晃60s。(3秒滴一滴)
6.沿管壁轻柔加入14ml 37℃预温的1640基础培养基终止(第1min加1ml，3min加3ml，最后缓慢加入10ml)。(匀速滴加)
7. 37℃静止5min后离心(800rpm，5min)，弃去上清(将离心管倾斜，吸去上清)。
8.将沉淀细胞轻轻用37℃预热的1640选择培养基重悬(不可吹打)，加入到事先准备好的预热培养基中后，混匀后滴加在上述含有滋养细胞的96孔培养板中，100μl/孔，置于37℃、5％CO2培养箱中培养，3-4d后半量换液，10d后改用HT培养基培养，2周后转用含10％FBS的1640培养基培养。
9.期间每天观察96孔板中克隆生长情况，一般在杂交瘤细胞布满孔 底1/10面积时，即可开始检测特异性抗体，筛选出所需要的杂交瘤细胞系。对于有特异性分泌抗体的细胞应及时克隆培养并冻存。通常需3-5次亚克隆才能获得稳定基因型和稳定分泌表型的细胞，且培养一段时间后需再次亚克隆。
实施例2 抗人B7-H4抗体重轻链可变区的测序
1.提取杂交瘤细胞的cDNA：从该杂交瘤细胞株中提取RNA，并使用RT-PCR技术，将获得的RNA反转录为cDNA；利用特定设计的上下游引物PCR克隆该杂交瘤细胞重链可变区(mVH)和轻链可变区(mVL)；
2.mVH和mVL分别与克隆载体(pJET cloning vector)连接，连接产物转化感受态细菌DH5a，由于pJET载体带有氨苄(Amp+)抗性基因，可将转化菌液涂在Amp抗性的LB固体培养基上，37°过夜培养；
3.待涂板细菌长出分散菌落，选择边缘清晰、生长良好的菌落，进一步测序鉴定；
4.根据测序结果保留候选的重轻链可变区序列，再次PCR扩增出与表达载体匹配的重链、轻链可变区序列，将PCR产物与双酶切预先处理的线性表达载体连接，连接产物转化感受态细菌DH5a，由于表达载体带有卡那霉素(Kana+)抗性基因，可将转化菌液涂在Kana抗性的LB固体培养基上，37°过夜培养；
5.测序方法参照4.对比两次测序结果，挑选正确序列的转化菌，扩大培养后进行质粒抽提；
6.连接有目的单抗重链和轻链可变区基因的表达载体共转染真核表达细胞株293。293细胞为悬浮培养，SFM4Transfx-293 without L-glutamine(liquid)无血清培养基传代扩增，转染时用
Figure PCTCN2018094692-appb-000004
FreeStyle TM293 Expression Medium无血清培养基替换。通过7天连续培养后收获上清，4000g离心30min，去除上清中细胞等杂质，并用0.45um滤器过滤除菌；
7.收获的上清含有目的抗体，Western blot检测表达单抗与转基因细胞CHO/B7-H4结合良好。
通过上述方法获得的抗人B7-H4抗体重链可变区：
Figure PCTCN2018094692-appb-000005
YYCARDYGSGAWYFDVWGAGTTVTVSS
三个高变区：GYSITSDFA、ISYSGDT、ARDYGSGAWYFDV。
抗体轻链可变区：
Figure PCTCN2018094692-appb-000006
CKQSYNLWTFGGGTKLEIK
三个高变区：QSLLNSRTRKNY、WAS、KQSYNLWT。
实施例3 杂交瘤细胞染色体核型分析
取生长良好的细胞，加入秋水仙素，使其终浓度为0.04～0.08μl/ml。培养2h后收集细胞(1000rpm，10min)于离心管中，逐滴加入0.5ml 37℃预温的0.075mol/L KCl溶液后，随即再补加5～10ml，用吸管轻轻吹打均匀，37℃孵育20min。在管中加入1ml新鲜配制的固定液(3份甲醇加1份冰醋酸，临用前配制)，1000rpm，离心10min弃上清。再加入8～10ml固定液，用吸管吹打均匀，固定15～20min，离心，弃上清。再加入5ml固定液，固定30min。离心弃上清，再加入1.5ml固定液，吹打均匀。取-10℃冰冻的载玻片，滴加1～2滴细胞悬液，用新鲜Giemsa溶液(1份Giemsa原液加9份0.075mol/L、pH6.8磷酸盐缓冲液)染色10～20min，流水冲洗后晾干。二甲苯透明三次，中性树脂封片。在显微镜下选择染色体分散良好、不重叠、无失散的标本，于油镜下观察记录并进行显微摄影。
实施例4 免疫组织化学技术
1.石蜡切片
取新鲜组织，修剪为1cm×1cm×0.3cm大小组织块，中性福尔马林 液中固定24h，梯度酒精溶液脱水(70％酒精15min、80％酒精15min、90％酒精1h、95％酒精2h，无水乙醇2h)，二甲苯透明(共两次，每次各30min)，浸蜡(共两次，第一次1h，第二次2h)，石蜡包埋后切5μm薄片，贴附于涂布多聚赖氨酸的玻片。
2.免疫组化染色
0.03％H 2O 2-甲醇孵育30分钟，滴加10％BSA，室温孵育30分钟，勿洗，滴加一抗(抗人CD105抗体稀释度为1：100)，以PBS代替一抗作为阴性对照，37℃孵育2h，PBS冲洗，滴加通用型二抗即用液，37℃孵育30min，PBS冲洗三遍，DAB-H2O2显色，经苏木素复染，1％盐酸-酒精分化，梯度乙醇中脱水(75％乙醇3min，95％乙醇I 3min，95％乙醇II 3min，100％无水乙醇I 3min，100％无水乙醇II 3min)后，吹风机吹干无水乙醇，中性树脂封片，显微镜下观察并拍照。

Claims (7)

  1. 一种抗人B7-H4单克隆抗体，包括重链和轻链，其特征在于，所述重链可变区(mVH)的氨基酸序列与SEQ ID NO.1相同，具体如下：DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLDWMGYISYSGDTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARDYGSGAWYFDVWGAGTTVTVSS；
    所述轻链可变区(mVL)的氨基酸序列与SEQ ID NO.2相同，具体如下：DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFILTISSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK。
  2. 如权利要求1所述的一种抗人B7-H4单克隆抗体，其特征在于，所述所述重链可变区(mVH)具有三个高变区为：GYSITSDFA、ISYSGDT、ARDYGSGAWYFDV。
  3. 如权利要求1所述的一种抗人B7-H4单克隆抗体，其特征在于，所述轻链可变区(mVL)具有三个高变区为：QSLLNSRTRKNY、WAS、KQSYNLWT。
  4. 权利要求1至3任一项权利要求所述的一种抗人B7-H4抗体重链和轻链可变区序列的鉴定方法，其特征在于，包括以下步骤：
    (1)B7-H4单克隆抗体的杂交瘤的制备；
    (1.1)免疫小鼠；
    (1.2)细胞培养；
    (1.3)融合与筛选；
    (2)B7-H4鼠/人嵌合抗体的制备；
    (2.1)提取杂交瘤细胞的cDNA：从该杂交瘤细胞株中提取RNA，并使用RT-PCR技术，将获得的RNA反转录为cDNA；
    利用特定设计的上下游引物PCR克隆该杂交瘤细胞的重链可变区(mVH)和轻链可变区(mVL)；所述特定设计的上下游引物的序列分别为核苷酸SEQ ID NO.3和核苷酸序列4；
    (2.2)所述重链可变区(mVH)和轻链可变区(mVL)分别与克隆载体(pJET cloning vector)连接，连接产物转化感受态细菌DH5a，由于pJET载体带有氨苄(Amp+)抗性基因，可将转化菌液涂在Amp抗性的LB固体培养基上，37°过夜培养；
    (2.3)待涂板细菌长出分散菌落，选择边缘清晰、生长良好的菌落，进一步测序鉴定；
    (2.4)根据测序结果保留候选的重链可变区(mVH)和轻链可变区(mVL)序列，再次PCR扩增出与表达载体匹配的重链可变区(mVH)和轻链可变区(mVL)序列，将PCR产物与双酶切预先处理的线性表达载体连接，连接产物转化感受态细菌DH5a，由于表达载体带有卡那霉素(Kana+)抗性基因，可将转化菌液涂在Kana抗性的LB固体培养基上，37°过夜培养；
    (2.5)测序方法参照(2.4)对比两次测序结果，挑选正确序列的转化菌，扩大培养后进行质粒抽提；
    (2.6)连接有目的单抗重链可变区(mVH)和轻链可变区(mVL)基因的表达载体共转染真核表达细胞株293；
    (2.7)收获的上清含有目的抗体，ELISA检测结果显示单抗4B2能较好地结合转基因细胞的B7-H4蛋白，证明上述重链和轻链可变区序列正确。
  5. 权利要求4所述的鉴定方法，其特征在于,步骤(2.6)中，所述真核表达细胞株293为悬浮培养，SFM4Transfx-293 without L-glutamine(liquid)无血清培养基传代扩增，转染时用
    Figure PCTCN2018094692-appb-100001
    FreeStyle TM293 Expression Medium无血清培养基替换；通过7天连续培养后收获上清，4000g离心30min，去除上清中细胞等杂质，并用0.45um滤器过滤除菌。
  6. 权利要求1至3任一项权利要求所述的一种抗人B7-H4单克隆抗体的应用，其特征在于，所述抗人B7-H4单克隆抗体用于制备用于肿瘤组织的免疫组化检测的试剂盒。
  7. 一种试剂盒，其特征在于，所述试剂盒包含权利要求1至3任 一项权利要求所述的抗人B7-H4单克隆抗体。
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