CN106084043B - One kind ZnT8 bispecific single chain antibody scFv-C22 Its Applications - Google Patents

One kind ZnT8 bispecific single chain antibody scFv-C22 Its Applications Download PDF


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CN106084043B CN201610520742.6A CN201610520742A CN106084043B CN 106084043 B CN106084043 B CN 106084043B CN 201610520742 A CN201610520742 A CN 201610520742A CN 106084043 B CN106084043 B CN 106084043B
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本发明公开了一种抗1型糖尿病ZnT8特异性单链抗体scFv‑C22及其应用。 The present invention discloses an anti-diabetic type 1 ZnT8 bispecific single chain antibody scFv-C22 and its application. 所述的抗1型糖尿病ZnT8特异性单链抗体的氨基酸序列如SEQ ID NO.5所示,其编码基因序列如SEQ ID NO.6所示。 ZnT8 amino acid sequence of the bispecific single chain antibody against type 1 diabetes, as shown in FIG SEQ ID NO.5, as its gene sequence SEQ ID NO.6. 本发明成功构建了T1D scFv噬菌体库,筛选并鉴定了ZnT8特异性scFv,该ZnT8特异性scFv可用于1型糖尿病诊断、治疗和/或预后判断试剂的制备。 The present invention successfully constructed TlD scFv phage library, screening and identification of specific scFv ZnT8, the specific scFv ZnT8 Diagnosis of Type 1 may be used to prepare and / or prognosis of the treatment agent.


一种ZnT8特异性单链抗体scFv-C22及其应用 One kind ZnT8 bispecific single chain antibody scFv-C22 Its Applications

技术领域 FIELD

[0001] 本发明属于生物医药技术领域,涉及一种抗1型糖尿病ZnT8特异性单链抗体ScFv-C22及其应用。 [0001] The present invention belongs to the field of biotechnology medicine, relates to an anti-diabetic type 1 ZnT8 bispecific single chain antibody ScFv-C22 and its application.

背景技术 Background technique

[0002] 锌转运体(ZnT)是一组将锌离子由胞内转运出胞外的跨膜蛋白,与ZIP家族蛋白共同维护细胞内外锌离子的平衡。 [0002] Zinc transporter (ZnT) is a group of the zinc ion transport from the extracellular intracellular transmembrane protein, the protein family with a common maintenance ZIP zinc ion balance inside and outside the cells. 2004年,Cheimienti等首次在ZnT家族中发现ZnT8蛋白,它由SLC30A8基因编码,高度特异的表达在胰腺中,后进一步研究发现ΖηΤ8特异的表达在胰岛β细胞上,在INS-I细胞中过表达ZnTS可促使锌离子的聚集并增强糖刺激后的胰岛素释放。 In 2004, Cheimienti other first discovered ZnT8 protein ZnT family, it is encoded by a gene SLC30A8, highly specific expression in the pancreas, after further study found ΖηΤ8 specific expression in islet β cells, through the INS-I cells expressing ZnTS zinc ions may promote aggregation and enhanced insulin release after glucose stimulation. 这些研究表明ZnTS在胰岛素的合成和分泌中发挥重要作用。 These studies indicate ZnTS play an important role in the synthesis and secretion of insulin.

[0003] 1型糖尿病(TlD)是由T淋巴细胞介导,以选择性破坏胰岛辟田胞为特征的器官特异性自身免疫性疾病。 [0003] Type 1 diabetes (TLD) are mediated by lymphocytes with T, selective destruction of islet cells is characterized by the provision of fields of organ-specific autoimmune diseases. 胰岛β细胞上存在着多种自身抗原,如胰岛素,谷氨酸脱羧酶(GAD),酪氨酸磷酸酶和ΖηΤ8。 There are many self-antigens, such as insulin, glutamic acid decarboxylase (of GAD), tyrosine phosphatase on pancreatic β cells and ΖηΤ8. 其中,ΖηΤ8被认为是TlDM的一个重要的自身抗原,Wenzlau等发现部分TlD高危患者发病前2年即可检测到ΖηΤ8自身抗体且长期维持在较高水平。 Which, ΖηΤ8 TlDM is considered to be an important autoantigen, Wenzlau found that high-risk patients before the onset of part TlD 2 years to detect autoantibodies to ΖηΤ8 and long-term at a higher level. 我们前期研究发现ΖηΤ8存在着多个抗原表位可被1型糖尿病患者自身反应性T细胞识别,且ΖηΤ8抗体水平与TlD的发展存在着显著相关性。 Our previous study found ΖηΤ8 there may be multiple epitopes of reactive T cells in type 1 diabetes itself, and ΖηΤ8 antibodies and the development of TlD there is a significant correlation. 早期检测ΖηΤ8抗体和ΖηΤ8特异性CD8+T细胞是预测和诊断TlD的重要手段。 Antibodies and ΖηΤ8 ΖηΤ8 specific CD8 + T cells is an important means for diagnosis and prediction of early detection TlD. 针对ΖηΤ8靶点的特异性治疗将成为阻止TlD进展的潜在方法。 For specific treatment ΖηΤ8 target will be a potential way to stop TlD progress.

[0004] 抗体研究迄今已有100多年历史,1975年Kohler和Mi Istein创立B淋巴细胞杂交瘤技术,鼠源单克隆抗体被广泛应用于疾病的诊断和治疗,但由于鼠源单克隆抗体对人体有免疫原性,易产生人抗鼠抗体反应(human anti-mouse antibody,HAMA和超敏反应,且由于其分子量较大,难以穿膜等缺点,限制了其在疾病治疗领域的应用。自20世纪八十年代中期,随着分子生物学的技术进展,国外学者建立了噬菌体抗体库技术。由于噬菌体表达的scFv抗体分子量较小,仅为完整抗体分子的六分之一,且具有较好的血管或组织屏障穿透性、半衰期短、结构稳定、不含Fc段,减少了与体内Fc受体结合而带来的不利影响等优势,已广泛地应用于分子生物学及免疫学、药理学等医学领域,是目前的研究热点。但目前尚未见抗1型糖尿病ZnT8特异性单链抗体的相关报道。 [0004] antibody research so far has been 100 years of history, 1975, Kohler and Mi Istein creation of B lymphocyte hybridoma technology, murine monoclonal antibodies are widely used in the diagnosis and treatment of disease, but because of murine monoclonal antibodies to the human body immunogenic, easy to produce human anti-mouse antibody response (human anti-mouse antibody, HAMA and hypersensitivity reactions, and because of their larger molecular weight, it is difficult to wear film disadvantage limits its application in the field of treatment of diseases since 20 mid-eighties, as technology advances in molecular biology, foreign scholars to establish a phage display technology. due to the small molecular weight antibody scFv phage expression, only one-sixth of an intact antibody molecule, and has good vessels or tissue barrier penetration, short half-life, stable structure, non-Fc fragment, brought reduced binding to the Fc receptors in vivo adverse effects and other advantages, it has been widely used in molecular biology and immunology, pharmacology medicine and other fields, is the current hot topic, but has yet to see the relevant reports ZnT8 specific single chain antibodies against type 1 diabetes.


[0005] 本发明的目的是针对现有技术的上述不足,提供一种抗1型糖尿病ZnTS特异性单链抗体。 Objective [0005] The present invention is the above deficiencies of the prior art, type 1 diabetes, there is provided a bispecific single chain antibody anti ZnTS.

[0006] 本发明的另一目的是提供该单链抗体的应用。 [0006] Another object of the present invention is to provide the use of a single chain antibody.

[0007] 本发明的目的可通过以下技术方案实现: [0007] The object of the present invention can be achieved by the following technical solution:

[0008] 抗1型糖尿病ΖηΤ8特异性单链抗体scFv_C22,包括轻链和重链, [0008] Type 1 diabetes, an anti-specific single chain antibodies ΖηΤ8 scFv_C22, including light and heavy chains,

[0009] 所述的轻链的可变区的氨基酸序列如SEQ ID NO. 1所示。 [0009] The amino acid sequence of the light chain variable region, such as SEQ ID NO. 1 FIG.

[0010] 所述的重链的可变区的氨基酸序列如SEQ ID NO.2所示; The amino acid sequence of the variable region of the heavy chain [0010] As shown in SEQ ID NO.2;

[0011] 所述的抗1型糖尿病ZnT8特异性单链抗体scFv-C22的氨基酸序列优选如SEQ ID NO. 5所示。 [0011] The anti-diabetic type 1 ZnT8 bispecific single chain antibody scFv-C22 preferably an amino acid sequence as SEQ ID NO. 5 FIG.

[0012] —种编码本发明所述的抗1型糖尿病ZnT8特异性单链抗体scFv-C22的基因,其中, [0012] - bispecific single chain antibody scFv-C22 gene encoding said species present invention against Type 1 diabetes of ZnT8, wherein

[0013] 编码轻链的可变区的核苷酸序列如SEQ ID NO.3所示。 The nucleotide sequence of the variable region [0013] encoding the light chain as shown in SEQ ID NO.3.

[0014] 编码重链的可变区的核苷酸序列如SEQ ID NO.4所示; [0014] The nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO.4;

[0015] 编码本发明所述的抗1型糖尿病ZnT8特异性单链抗体scFv-C22的基因的核苷酸序列优选如SEQ ID NO.6所示。 [0015] Type 1 diabetes, the anti-encoding the present ZnT8 bispecific single chain antibody scFv-C22 gene is preferably a nucleotide sequence as shown in SEQ ID NO.6.

[0016] 本发明所述的抗1型糖尿病ZnT8特异性单链抗体scFv-C22在制备1型糖尿病诊断、 治疗和/或预后判断试剂中的应用。 [0016] Antibodies and / or prognostic agents in the present invention is applied against type 1 diabetes ZnT8 specific single chain scFv-C22 Diagnosis of Type 1 in the manufacture, treatment.

[0017] 本发明所述的编码编码权利要求1所述的抗1型糖尿病ΖηΤ8特异性单链抗体scFv-C22的基因在制备1型糖尿病诊断、治疗和/或预后判断试剂中的应用。 [0017] The coding of the present invention as claimed an anti-diabetic type 1 gene of claim 1 ΖηΤ8 bispecific single chain antibody scFv-C22 in the preparation of diagnosis of type 1 diabetes, and / or prognostic reagent application requirements treatment.

[0018] 有益效果: [0018] beneficial effects:

[0019] 本发明首次从TlD病人外周血基因中构建了一个库容达IX IO8的scFv噬菌体抗体库。 [0019] The invention firstly constructs a storage capacity of IX IO8 scFv phage antibody library from the peripheral blood of patients in gene TlD. 我们招募了50例初发的TlD患者以保证库的特异性、多样性和代表性。 We recruited 50 cases of patients with newly diagnosed TlD to ensure specificity, diversity and representation of libraries. 从该库中,我们可以筛选TID的各种自身抗体。 From the library, we can filter all kinds of TID autoantibodies.

[0020] ZnT8抗原是TlD的重要自身抗原,具有六次跨膜结构,它由SCL30A8基因编码,定位于人第8号染色体q24.11上,包含8个外显子,编码369个氨基酸。 [0020] ZnT8 TlD antigen is an important autoantigen, having six transmembrane, encoded by a gene that SCL30A8, located on human chromosome 8 q24.11, containing 8 exons, encoding 369 amino acids. ΖηΤ8自身抗体在TlD的发生发展中发挥了重要作用。 ΖηΤ8 autoantibodies play an important role in the development of TlD. 目前,ΖηΤ8蛋白的氨基末端(l-74aa)和羧基末端(268-369aa)被认为是自身反应性T细胞识别的主要区域。 Currently, the amino terminus of the protein ΖηΤ8 (l-74aa) and carboxy-terminal (268-369aa) is considered to be the main area of ​​their cell recognition reactive T. 由于ZnTS蛋白影响着胰岛素的合成与分泌,所以对其抗体作用机制的深了解对治疗TlD具有重大意义。 Since ZnTS affect protein synthesis and secretion of insulin, it is of great significance for the treatment of TlD its deep understanding of the mechanisms of antibody action. 本发明成功筛选了ZnTS特异性scFv。 The present invention successfully screened ZnTS specific scFv. 为保证筛选结果的多样性,我们选择了ZnTS蛋白的氨基羧基融合肽进行scFv的筛选。 To ensure the diversity of screening results, we selected amino carboxyl ZnTS protein fusion peptide screening of scFv. 已经有大量报道发现TlD患者中ZnT8抗原325位氨基酸存在着点突变,由精氨酸(Arg)突变为酪氨酸(Trρ),该位点突变与ZnT8抗体的识别密切相关,ZnT8羧基末端的突变二聚体(Arg325Trp)对检测ZnT8抗体具有高度的特异性和敏感性。 Number of reports have been found in patients with TlD ZnT8 antigenic amino acid 325 there is a point mutation from arginine (Arg) mutated to tyrosine (Trρ), it is closely related to the mutation site and the identification ZnT8 antibodies, the carboxyl terminus ZnT8 mutant dimer (Arg325Trp) is highly specific and sensitive detection of antibodies ZnT8. 所以我们进一步使用ZnT8羧基末端的二聚体(Arg325Trp)鉴定了scFv,结果表明scFv-C22能与该二聚体结合。 Therefore, we further use dimer carboxy terminus ZnT8 (Arg325Trp) identified scFv, scFv-C22 results show that the dimer can bind. 免疫组化结果进一步显示scFv能特异性识别胰岛细胞,具有良好的生物学活性。 Immunohistochemistry results further show that scFv specifically recognize pancreatic islet cells, having good biological activity.

[0021] 本发明成功构建了TlD scFv噬菌体库,筛选并鉴定了ZnT8特异性scFv,该ZnT8特异性scFv可用于1型糖尿病诊断、治疗和/或预后判断试剂的制备。 [0021] The present invention successfully constructed a TLD scFv phage library, screening and identification of specific scFv ZnT8, the specific scFv ZnT8 Diagnosis of Type 1 may be used to prepare and / or prognosis of the treatment agent.


[0022] 图1.重组ΖηΤ8基因构建.Μ:核酸标准品;泳道1:ΖηΤ8氨基末端基因(l-74aa)约250bp;泳道2:ZnT8羧基末端基因(268-369aa)约350bp;泳道3:ZnT8氨基羧基融合基因(1-74aa,268-369aa)约550bp;泳道4:ΖηΤ8羧基端二聚突变体基因(Arg325Trp)约650bp。 [0022] Figure 1. Construction of a recombinant gene ΖηΤ8 .Μ: nucleic acid standard; Lane 1: ΖηΤ8 amino terminus of the gene (l-74aa) of about 250bp; Lane 2: ZnT8 carboxy terminus of the gene (268-369aa) about 350bp; Lane 3: ZnT8 aminocarboxy fusion gene (1-74aa, 268-369aa) about 550bp; lane 4: ΖηΤ8 carboxy terminus mutant gene dimer (Arg325Trp) about 650bp. 图2. 重组ZnT8蛋白的表达纯化及鉴定. Figure 2. Expression and Purification of Recombinant Protein ZnT8.

[0023] Α:ΖηΤ8氨基羧基融合蛋白的Western blotting检测(泳道1:蛋白纯化前;泳道2: 纯化后的蛋白;泳道3:流穿);B: ZnT8氨基羧基融合蛋白的SDS-PAGE检测(M:蛋白标准品;泳道1:蛋白纯化前;泳道2:纯化后的蛋白;泳道3:流穿);C: ZnTS羧基端二聚突变体蛋白Western blotting检测(泳道1:蛋白纯化前;泳道2:纯化后的蛋白;泳道3:流穿);D:ZnT8羧基端二聚突变体蛋白SDS-PAGE检测(M:蛋白标准品;泳道1:蛋白纯化前;泳道2:纯化后的蛋白;泳道3:流穿)。 [0023] Α: ΖηΤ8 aminocarboxy by Western blotting of the fusion protein (Lane 1: pre-protein purified; Lane 2: purified protein; lane 3: flow through); B: ZnT8 aminocarboxy SDS-PAGE detection of the fusion protein ( M: protein standards; lane 1: pre-protein purified; lane 2: purified protein; lane 3: flow through); C: ZnTS carboxy terminus dimeric mutant proteins by Western blotting (lane 1: before protein purification; lane 2: purified protein; lane 3: flow through); D: ZnT8 carboxy-terminated dimer mutant proteins by SDS-PAGE (M: protein standards; lane 1: pre-protein purified; lane 2: purified protein; lane 3: flow-through).

[0024] ZnT8氨基羧基融合蛋白和羧基端二聚突变体蛋白分子量分别约20kD和25kD。 [0024] ZnT8 amino and carboxy-terminal carboxy fusion protein dimer mutant protein of molecular weight of approximately 20kD and 25kD.

[0025] 图3 .ELISA检测氨基羧基融合蛋白与商品化ZnT8抗体的结合能力。 [0025] FIG. 3 .ELISA detection aminocarboxy fusion protein binding capacity and commercialization ZnT8 antibody.

[0026] 图4.T1D scFv基因扩增.Μ:核酸标准品;泳道I=VAPCR产物(〜350bp);泳道2:Vk PCI^*^^J(〜350bp)J1dt3:VHPCI^*^^J(〜450bp)J1dt4:scFvPCI^*^^J(〜800bp)。 [0026] FIG 4.T1D scFv gene amplification .Μ: nucleic acid standard; Lane I = VAPCR product (~350bp); Lane 2: Vk PCI ^ * ^^ J (~350bp) J1dt3: VHPCI ^ * ^^ J (~450bp) J1dt4: scFvPCI ^ * ^^ J (~800bp).

[0027] 图5.scFv-C22的纯化及鉴定· [0027] FIG. Purification and identification of · 5.scFv-C22

[0028] A. SDS-PAGE检测scFv (M:蛋白标准品;泳道1: scFv-C22纯化前;泳道2 :纯化后的scFv_C22;泳道3:scFv_C22流穿。);B.Western blotting analyze检测scFv (泳道l:scFv_ C22纯化前;泳道2:纯化后的scFv-C22;泳道3: scFv-C22流穿)。 [0028] A. SDS-PAGE detection of scFv (M: protein standards; Lane 1: scFv-C22 before purification; Lane 2: purified scFv_C22; Lane 3:. ScFv_C22 flowthrough); B.Western blotting analyze detected scFv (lane l: purified before scFv_ C22; lane 2: scFv-C22 purified; lane 3: scFv-C22 flow-through).

[0029] 图6.Western blotting检测scFv与ZnT8氨基羧基融合蛋白的结合能力. [0029] FIG 6.Western blotting and detection of scFv binding ability ZnT8 aminocarboxy fusion protein.

[0030] 泳道1: scFv-C22检测ΖηΤ8氨基羧基融合蛋白;泳道2:阴性对照。 [0030] Lane 1: scFv-C22 fusion protein was detected ΖηΤ8 aminocarboxy; Lane 2: negative control.

[0031] 图7. SCFV-C22与ΖηΤ8氨基羧基融合蛋白的亲和力检测。 [0031] FIG. 7. SCFV-C22 fusion protein was detected with affinity ΖηΤ8 amino carboxyl group.

[0032] 图8』1^3厶检测8〇?¥-022对21^8羧基端二聚突变体蛋白的结合能力。 [0032] FIG. 8 "1 ^ 3 Si detector 8〇? ¥ 21 to ^ 8-carboxy -022 binding capacity dimeric mutant proteins ends.

[0033] 图9.免疫组化检测scFv抗体对人胰腺组织的识别能力 [0033] Figure 9. Immunohistochemical detection of scFv antibody recognition ability of human pancreatic tissue

[0034] A:胰岛细胞HE染色结果;B: C22检测胰岛细胞结果。 [0034] A: HE staining of islet cells; B: C22 islet cell detection result. 行1:放大40倍;行2 :放大200 倍· Line 1: x40; line 2: 200 times magnification ·

具体实施方式 Detailed ways

[0035] 实施例1重组ZnT8原核表达质粒的构建与表达 [0035] Construction and Expression ZnT8 prokaryotic expression of recombinant plasmid Example 1

[0036] 设计引物ZF和LR (表1),以ΖηΤ8质粒(购自北京义翘神州生物技术有限公司, HG11621-M)为模板,扩增人ΖηΤ8氨基末端(l-74aa)基因,设计引物LF和CR (表1),以ΖηΤ8质粒为模板,扩增人ΖηΤ8羧基末端(269-368aa)基因,,PCR产物胶回收后用引物ZF和CR通过overlap构建氨基羧基融合基因。 [0036] Primers were designed ZF and LR (Table 1), to ΖηΤ8 plasmid (available from Beijing Sino Biological Technology Co., HG11621-M) as a template, to amplify human ΖηΤ8 amino terminus (l-74aa) gene, primers were designed LF and CR (table 1), to ΖηΤ8 plasmid as a template, amplification of carboxyl terminus of human ΖηΤ8 (269-368aa) Construction of the fusion gene by overlap aminocarboxy gene ,, PCR products after gel and recovered using primers ZF CR.

[0037] 参考US9023984 (B2)序列表中的SEQ ID如.55设计21^8羧基末端二聚突变体基因,如SEQ ID No. 7所示,并委托生物公司合成,该二聚体含2个人ZnT8羧基末端基因,第一个在325为氨基酸为精氨酸(Arg),第二个在325位为酪氨酸(Trp),该二聚体中2个人ΖηΤ8羧基末端基因通过一段柔性肽链接。 [0037] Reference US9023984 (B2) in the Sequence Listing as SEQ ID 21 ^ 8 .55 design dimer carboxy terminus mutant genes, such as SEQ ID No. 7, and the company entrusted biological synthesis, the dimer containing 2 personal ZnT8 carboxy terminal sequence in the first 325 amino acids is arginine (Arg), at the second tyrosine 325 (Trp), the dimer personal ΖηΤ8 2 gene by a length of flexible carboxy terminus peptide link. 设计引物CF和CR用于扩增ZnTS羧基端二聚突变体基因。 CF and CR primers designed for amplification of the carboxy terminal dimer ZnTS mutant gene.

[0038] 表1.引物序列 [0038] Table 1. Primer sequences

[0039] [0039]

Figure CN106084043BD00051

[0040] 引物ZF和LR用于扩增ZnT8氨基末端基因(l-74aa),引物LF和CR用于扩增ZnT8羧基末端基因(268-369aa),引物ZF和CR用于overlap扩增ΖηΤ8氨基羧基融合基因,引物CF和CR 用于扩增ZnTS羧基端二聚突变体基因。 [0040] The primers used to amplify the LR ZF ZnT8 amino terminus gene (l-74aa), CR and LF primer for amplification of carboxyl terminus ZnT8 gene (268-369aa), and CR ZF primer for amplification of overlap amino ΖηΤ8 carboxy fusion gene, the primers CF and CR are used to amplify the carboxy terminal dimer ZnTS mutant gene.

[0041] 上述两个重组基因分别经SacI和XbaI双酶切后插入pcold Π质粒中。 [0041] The two recombinant genes, respectively, after insertion SacI and XbaI digested plasmid pcold Π. 连接产物转化入大肠杆菌BL21 (DE3)培养后挑选阳性克隆进行测序。 After ligation product was transformed into E. coli BL21 (DE3) culture positive clones were sequenced. 将测序正确的克隆在LB培养基中37°C扩增至D (600)值达1.0后加入IPTG诱导剂至终浓度为ImM/L,15°C诱导表达2处。 The correct sequence of the clones in LB medium to amplify 37 ° C D (600) value of 1.0 after addition of IPTG inducer to the final concentration of ImM / L, 15 ° C at 2 expression induced. 4°(:,10 000 Xg离心后收集菌体-80°C保存。 4 ° (:, 10 000 Xg harvested by centrifugation after stored -80 ° C.

[0042] 将纯化的人ZnT8氨基羧基融合蛋白包被ELISA板,从1.6ug/孔倍比稀释至0. Iug/ 孔,检测融合蛋白与商品化的ZnTS抗体结合的量效变化。 [0042] Purified human ZnT8 aminocarboxy ELISA plates were coated with the fusion protein, were diluted from 1.6ug / times larger than the hole to 0. Iug / aperture detection antibody fusion protein dose variation ZnTS binding and commercialization.

[0043] ZnT8氨基羧基融合基因约550bp,羧基二聚突变体基因约650bp,PCR产物电泳结果见图1,均成功插入pcold Π载体,测序结果与理论相符。 [0043] ZnT8 about 550bp fused gene aminocarboxy, carboxy dimeric mutant gene about 650bp, PCR products were electrophoresed results shown in Figure 1, were successfully inserted into the vector pcold Π, consistent with the theoretical sequence.

[0044] 抗原经表达纯化复性后电泳结果见图2,可见纯化后ZnTS氨基羧基融合肽分子量约20kD,羧基二聚突变体分子量约25kD。 After [0044] Antigen Expression Purification, renaturation by electrophoresis results shown in Figure 2, seen after purification ZnTS aminocarboxy fusion peptide molecular weight of about 2OkD, carboxy mutant dimer molecular weight of about 25kD.

[0045] ELISA检测显示ZnT8氨基羧基融合蛋白能与商品化的ZnT8抗体特异性结合,可用于ΖηΤ8特异性scFv的筛选(图3)。 [0045] ELISA analysis showed ZnT8 aminocarboxy fusion protein specifically binds to an antibody ZnT8 commercially available, can be used to screen ΖηΤ8 specific scFv (FIG. 3).

[0046] 实施例2T1D scFv噬菌体抗体库的构建 [0046] Example 2T1D scFv Phage Display Library Construction

[0047] 实验标本为50位初发的TlD志愿者的外周血。 [0047] The experimental specimens of the peripheral blood of newly diagnosed TlD 50 volunteers. 其中31位志愿者血清ZnT8抗体阳性。 ZnT8 31 volunteers serum antibody positive. 血清ΖηΤ8抗体水平通过放射免疫配体法进行检测(详细方法参见Gu Y,Zhang M,Chen Η, Wang Z1Xing C,Yang H,et al.Discordant association of islet autoantibodies with high-risk hla genes in Chinese type ldiabetes·Diabetes/metabolism research and reviews 2011;27:899-905)。 Serum antibody levels were detected ΖηΤ8 (For details, see Gu Y, Zhang M, Chen Η, Wang Z1Xing C, Yang H, et al.Discordant association of islet autoantibodies with high-risk hla genes in Chinese type ldiabetes by radioimmunoassay method ligand · Diabetes / metabolism research and reviews 2011; 27: 899-905). 所有患者均签署知情同意书。 All patients gave written informed consent. 分选TlD志愿者外周血TOMC。 Sorting TlD volunteers. TOMC. 提取TlD志愿者外周血PBMC RNA,接着反转录合成cDNA,具体操作步骤按说明书进行。 Extraction TlD peripheral blood of volunteers PBMC RNA, followed by reverse transcription cDNA, specific steps according to instructions. 通过RT-PCR方法,用人源单链抗体(scFv)通用引物(表2)扩增重链和轻链,通过overlap PCR链接成scFv,经Sfi I酶切后插入pcomb3XSS质粒(购自BioVector NTCC保藏中心)中。 By RT-PCR method, employing single-chain antibody (scFv) universal primers (Table 2) amplify the heavy and light chains, linked by overlap PCR as scFv, Sfi by insertion pcomb3XSS plasmid (available from I digested deposited BioVector NTCC Center) in. 连接产物经过多次电转化入大肠杆菌XLl-Blue (购自Stratagene ,Cat .#200228),用SB培养基扩大培养后加辅助噬菌体VCSM13超感染。 After several rounds of ligation product was transformed into E. coli XLl-Blue (available from Stratagene, Cat. # 200228), SB medium plus expansion was VCSM13 helper phage superinfection. 次日收集上清经PEG-8000/NaCl沉淀后获得TlD scFv噬菌体抗体库。 The next day the supernatant was collected after the PEG-8000 / NaCl precipitate obtained TlD scFv phage antibody library.

[0048] 表2.人源scFv通用引物序列 [0048] Table 2. humanized scFv universal primer sequence

Figure CN106084043BD00061

Figure CN106084043BD00071

Figure CN106084043BD00081

[0058]上述经RT-PCR后成功扩增抗体重链及轻链,大小约400bp,单链抗体基因条带在750bp左右(图4),胶回收后插入pcomb3XSS载体中,电转化入大肠杆菌XLl-Blue,经VCSM13 超感染后获得噬菌体抗体库,经检测库容为IX 1〇8。 [0058] After the above was amplified by RT-PCR of antibody heavy and light chains, the size of about 400bp, single-chain antibody gene strip around 750bp (FIG. 4), is inserted into a vector pcomb3XSS After gel purification, transformed into E. coli electrical XLl-Blue, was obtained VCSM13 phage library after superinfection, IX 1〇8 detected storage capacity.

[0059] I · 2 · 4ZnT8特异性scFv的筛选 [0059] I · 2 · 4ZnT8 specific scFv screening

[0060] 将纯化的ZnT8氨基羧基融合蛋白按lyg/孔包被ELISA板,经过4轮的“吸附”、“洗脱”、“扩增”后(Zhang X,Qi X,Zhang Q,Zeng X,Shi Z,Jin Q,et al.Human 4f5single_ chain fv antibody recognizing a conserved halepitope has broad neutralizing potency against h5nl influenza a viruses of different clades.AntiviraI research 2013;99:91-9),phage-ELISA进行检测。 After [0060] The purified fusion proteins were ZnT8 aminocarboxy lyg / hole ELISA plates were coated, after four rounds of "adsorption", "washout", "amplification" (Zhang X, Qi X, Zhang Q, Zeng X , Shi Z, Jin Q, et al.Human 4f5single_ chain fv antibody recognizing a conserved halepitope has broad neutralizing potency against h5nl influenza a viruses of different clades.AntiviraI research 2013; 99: 91-9), phage-ELISA for detection. 挑选OD值高的阳性克隆提取质粒并送测序,测序结果与人类免疫球蛋白序列(IMGT数据库)(Ehrenmann F,Kaas Q,Lefranc MP.Imgt/3dstructure~db and imgt/domaingapalign:A database and a tool for immunoglobulins or antibodies ,T cell receptors,mhc,igsf and mhcsf.Nucleic acids research 2010;38:D301-7.Lefranc MPjGiudicelli V1Duroux P1Jabado-Michaloud J1Folch G1Aouinti S,et al.Imgt (r), the international immunogenetics information system (r) 25years on.Nucleic acids research 2015;43:D413_22.)进行比对并进行CDR区的划分。 Selected high OD value of positive clone was extracted and sent to sequencing, the sequencing results with human immunoglobulin sequences (IMGT Database) (Ehrenmann F, Kaas Q, Lefranc MP.Imgt / 3dstructure ~ db and imgt / domaingapalign: A database and a tool for immunoglobulins or antibodies, T cell receptors, mhc, igsf and mhcsf.Nucleic acids research 2010; 38: D301-7.Lefranc MPjGiudicelli V1Duroux P1Jabado-Michaloud J1Folch G1Aouinti S, et al.Imgt (r), the international immunogenetics information system ( r) 25years on.Nucleic acids research 2015; 43: D413_22) to compare and divide CDR regions.

[0061] 经过4轮的筛选,ZnT8特异性scFv得以富集。 [0061] After the screening of four, ZnT8 specific scFv to enrichment. 每一轮噬菌体滴度见表3。 Each round of phage titers are shown in Table 3. 将最后一轮洗脱的噬菌体感染大肠杆菌XLl-Blue后,随机选择160个克隆。 After the last round of the eluted phage infection of E. coli XLl-Blue, 160 randomly selected clones. Phage ELISA结果显示有23 个阳性克隆,其中有11个克隆㈤值较高,经测序得到7株scFv。 Phage ELISA results showed 23 positive clones, 11 clones which have a higher value (v), were selected and sequenced seven scFv. 我们对这7株scFv进行了⑶R 区的划分,均符合人源scFv抗体特征。 We scFv these seven were divided ⑶R area, are in line with human scFv antibody characteristics. 其中,scFv C27和C220D值最高,因此我们选择scFv-C22进行了进一步研究。 Which, scFv C27, and C220D highest value, so we choose scFv-C22 were further studied.

[0062] 表3. ZnT8特异性噬菌体抗体的富集 [0062] Table 3. ZnT8 enrichment of specific phage antibodies

Figure CN106084043BD00091

[0064] 得率(%)=洗脱后的噬菌体密度(pfu) X 100/吸附前的噬菌体密度(pfu) [0064] Yield (%) = Density phage (PFU) of phage were eluted after density (PFU) before X 100 / Adsorption

[0065] 将测序正确的阳性克隆转化入大肠杆菌ToplOF',挑单克隆菌落接种于含20mM MgCl2的SB培养基中,37°C振摇8h后加入IPTG至终浓度lmM,37°C诱导表达2处。 [0065] A positive clone with correct sequence was transformed into E. coli ToplOF ', pick monoclonal colonies were inoculated in SB medium containing 20mM MgCl2 was added to a final concentration of lmM IPTG after shaking 37 ° C 8h, 37 ° C induced expression at 2. 4°(:,10 000 Xg离心后收集菌体_80°C保存。 4 ° (:, 10 000 Xg harvested by centrifugation after storage _80 ° C.

[0066] 细菌沉淀用含8M尿素的磷酸盐缓冲液重悬,置于冰上,用超声破碎细胞仪进行裂解。 [0066] The bacterial pellet was resuspended in phosphate buffer containing 8M urea, placed on ice, cells were disrupted with an ultrasonic instrument for lysis. 10000 X g 4°C离心30min,上清经0.22μηι滤膜过滤后用HisTrap亲和层析柱纯化蛋白。 10000 X g 4 ° C centrifugation 30min, the supernatant was filtered after 0.22μηι membrane protein was purified by affinity chromatography HisTrap. 洗脱下的目的蛋白通过浓度梯度透析复性至尿素小于〇. 125mM,超滤浓缩后得到高浓度蛋白。 The protein eluted by the concentration gradient dialyzed billion to less than urea. 125mM, high protein concentration after ultrafiltration was concentrated. Western与SDS-PAGE结果表明我们成功表达并纯化了scFv-C22,其分子量约30kD (图5) SDS-PAGE and Western results showed that we have successfully expressed and purified scFv-C22, having a molecular weight of about a 30 kD (FIG. 5)

[0067] 实施例3ZnT8特异性scFv的特性分析 [0067] Example characteristic specific scFv embodiment 3ZnT8 Analysis

[0068] Western blot检测:将纯化的重组ZnT8氨基羧基融合蛋白经12%蛋白质胶电泳后转至PVDF膜上,5 %牛奶封闭后,用纯化的scFv作为一抗,HRP标记的HA抗体作为二抗,检测scFv-C22与ΖηΤ8的结合活性。 [0068] Western blot detection: purified recombinant fusion protein ZnT8 aminocarboxy 12% after gel electrophoresis of proteins onto PVDF membrane, blocked after 5% milk, as with purified scFv antibody, the HRP as a secondary antibody labeled HA antibody detection and binding activity of scFv-C22 ΖηΤ8 of. 结果显示scFv-C22可检测到氨基羧基融合蛋白,在20kD左右可见明显条带,与预期相符,而阴性对照未见条带,说明scFv-C22与ZnTS蛋白具有良好的结合能力(图6)。 The results showed that scFv-C22 detectable amino carboxy fusion protein, clearly visible band of about 2OkD, as expected, no bands while the negative control, indicating that scFv-C22 ZnTS with good protein binding capacity (FIG. 6).

[0069] 亲和力检测:由苏州百拓生物技术有限公司完成。 [0069] The affinity of test: By Ltd. Suzhou 100 Extension biotechnology. 亲和力结果显示scFv-C22与ΖηΤ8氨基羧基融合蛋白具有较高的亲和力,亲和力为5.953 Xl(T\D (M)(图7)。 The results show the affinity of the scFv-C22 aminocarboxylic ΖηΤ8 fusion protein has a higher affinity, affinity of 5.953 Xl (T \ D (M) (FIG. 7).

[0070] ELISA检测:将纯化的ZnT8羧基末端二聚突变体蛋白按0.5yg/孔包被ELISA板, scFv-C22从1:10倍比稀释至1:20480,检测8〇?¥-022与21^8结合的量效变化。 [0070] ELISA Detection: ZnT8 carboxy terminus of the purified mutant proteins were dimeric 0.5yg / hole ELISA plates were coated, scFv-C22 serial dilutions from 1:10 to 1: 20480, and detecting 8〇 ¥ -022? 21 ^ 8 changes the amount of binding effect. 如图8所示,随着scFv-C22浓度的倍比稀释,其与ZnTS羧基端二聚突变体蛋白的结合能力逐渐降低,说明scFv-C22以浓度依赖方式与ZnT8抗原肽结合。 As shown in FIG binding capacity, as serial dilutions of scFv-C22 concentration, with the carboxy terminal 8 ZnTS dimeric mutant proteins gradually decreases, indicating scFv-C22 concentration-dependent manner ZnT8 binding antigenic peptides.

[0071] 免疫组化:我们将C22抗体作为一抗,通过间接标记的方法进行免疫组化实验,抗体孵育后的人胰腺组织切片的胰岛细胞呈强阳性,说明C22抗体具有良好的生物活性,能够特异的识别人胰岛辟田胞表达的ΖηΤ8蛋白。 [0071] Immunohistochemistry: We C22 antibody as the primary antibody, immunohistochemistry experiments by the method of indirect labeling, human after antibody incubations pancreatic tissue sections of islet cells were strongly positive, indicating that C22 antibody has a good biological activity, capable of specifically recognizing human pancreatic islets of provision ΖηΤ8 expression of cell fields.

[0072] 以上结果均表明scFv_C22能够与人ΖηΤ8特异性结合。 [0072] These results show that scFv_C22 capable of binding to human ΖηΤ8 specificity.

Claims (2)

1. ZnT8特异性单链抗体scFv-C22,其特征在于: 轻链的可变区的氨基酸序列如SEQ ID NO. 1所示; 重链的可变区的氨基酸序列如SEQ ID NO.2所示; 所述的ZnT8特异性单链抗体SCFV-C22的氨基酸序列如SEQIDN0.5所示。 1. ZnT8 bispecific single chain antibody scFv-C22, wherein: the amino acid sequence of the variable light chain region as shown in SEQ ID NO 1; the amino acid sequence of the variable region of the heavy chain such as SEQ ID NO.2 shown; ZnT8 the specific antibody amino acid sequence of SCFV-C22 single-chain as shown SEQIDN0.5.
2. —种编码权利要求1所述的ZnT8特异性单链抗体scFv-C22的基因,其特征在于: 编码轻链的可变区的核苷酸序列如SEQ ID NO.3所示; 编码重链的可变区的核苷酸序列如SEQ ID NO.4所示; 其核苷酸序列如SEQ ID NO.6所示。 2. - ZnT8 bispecific single chain scFv-C22 gene according to claim 1 antibody germline encoded, wherein: the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO.3; encoding the heavy chain variable region nucleotide sequence as shown in SEQ ID NO.4; nucleotide sequence shown as SEQ ID NO.6.
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