树鼩VASN单克隆抗体的制备及其应用探索

doi:10.12300/j.issn.1674-5817.2025.027

摘要/Abstract

摘要：

目的通过表达和纯化树鼩Vasorin（VASN）重组蛋白，免疫小鼠制备抗树鼩VASN单克隆抗体并探讨抗体的应用。方法用反转录PCR（Reverse Transcription-Polymerase Chain Reaction，RT-PCR）法体外扩增树鼩Vasn基因全长序列，将树鼩Vasn基因插入pET30a载体中，构建树鼩pET30a-VASN重组载体，对重组质粒用BamHI酶和SalI酶进行双酶切和测序鉴定；将测序正确的重组载体转化至大肠杆菌BL21（DE3），异丙基-β-D-硫代半乳糖苷（Isopropyl β-D-thiogalactoside，IPTG）诱导表达树鼩VASN重组蛋白；通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis，SDS-PAGE）电泳分离蛋白，氯化钾（Potassium chloride，KCl）切胶纯化树鼩VASN重组蛋白；将纯化的树鼩VASN重组蛋白对BALB/c小鼠进行4次免疫，酶联免疫吸附实验（Enzyme Linked Immunosorbent Assay，ELISA）检测小鼠血清效价；取效价达到1：10 000以上的小鼠脾脏细胞进行细胞融合，通过含黄嘌呤、氨基蝶呤及胸腺嘧啶核苷（Hypoxanthine-Aminopterin-Thymidine，HAT）的培养基筛选杂交瘤细胞，ELISA法筛选出能够分泌特异性抗体的阳性杂交瘤细胞株，并通过有限稀释法获得单克隆细胞株；通过腹水诱生法大量制备VASN单克隆抗体并利用rProtein G纯化抗体，ELISA、实时定量反转录PCR （Real-Time Quantitative Reverse Transcription PCR，qRT-PCR）、Western blotting检测单克隆抗体的亲和力和特异性，以及树鼩永生化成纤维细胞和树鼩组织中VASN的表达情况。结果成功扩增出树鼩Vasn基因全长序列并构建了树鼩pET30a-VASN重组质粒载体，重组质粒载体经测序得到的序列与树鼩Vasn目的基因序列一致；重组载体表达后的蛋白主要以包涵体的形式存在，重组树鼩VASN蛋白纯化后的纯度达到90%，符合后续免疫实验的要求；利用重组树鼩VASN蛋白4次免疫小鼠后，小鼠血清抗体效价达1：729 000；利用杂交瘤技术及有限稀释法获得单克隆阳性杂交瘤细胞株，通过腹水诱生法及纯化后得到的单克隆抗体，ELISA法测定单克隆抗体的亲和力常数值达2.59×10⁷mol/L；Western blotting检测单克隆抗体特异性，结果显示，树鼩VASN单克隆抗体能与树鼩VASN重组蛋白结合，而与猪视黄醇结合蛋白4（Retinol-Binding Protein 4，RBP4）重组蛋白、人源VASN-富含亮氨酸重复序列（Leucine Rich Repeat，LRR）重组蛋白以及牛血清白蛋白（Bovine Serum Albumin，BSA）标准蛋白均无结合反应；Western blotting检测树鼩各主要组织VASN表达，结果显示抗树鼩VASN单抗能与树鼩的主要器官组织（心脏、肝脏、脾脏、肺脏、肾脏、肌肉）在相对分子量约70 000位置出现特异性的条带且背景清晰；qRT-PCR检测结果显示树鼩脾脏组织和肺脏组织Vasn的mRNA表达水平相对较高，利用树鼩VASN单克隆抗体进行免疫组化检测，发现该基因mRNA水平表达较高的树鼩脾脏组织、肺脏组织、树鼩永生化成纤维细胞中免疫组化呈现阳性结果，在相对低表达的组织中呈现阴性结果。结论成功制备了抗树鼩VASN的单克隆抗体，该抗体可用于树鼩永生化成纤维细胞、树鼩脾脏组织和肺脏组织免疫组化检测，研究结果为进一步探索VASN在树鼩模型中功能研究提供了重要的工具。

关键词: Vasorin（VASN）, 树鼩, 单克隆抗体

Abstract:

Objective To prepare monoclonal antibody against tree shrew Vasorin (VASN) by expressing and purifying the recombinant protein of tree shrew VASN and immunizing mice, and to explore the application of antibody. Methods The full-length sequence of tree shrew Vasn gene was amplified in vitro by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and the tree shrew Vasn gene was inserted into pET30a vector to construct tree shrew pET30a-VASN recombinant vector. The recombinant plasmid was identified by double enzyme cleavage and sequencing with BamHI enzyme and SalI enzyme. The recombinant vector with correct sequencing was transformed into Escherichia coli BL21 (DE3), and the recombinant protein was expressed in E. coli BL21 (DE3) through Isopropyl β-D-thiogalactoside (IPTG) induction. The protein was isolated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis, and the recombinant protein of VASN was purified by Potassium chloride (KCl) gel. BALB/c mice were immunized with purified tree shrew VASN recombinant protein for four times, and serum titer was detected by Enzyme Linked Immunosorbent Assay (ELISA). Mouse spleen cells with titer of more than 1:10 000 were selected for cell fusion, Hypoxanthine-Aminopterin-Thymidine (HAT) culture medium was used to screen hybridoma cells, ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies, and monoclonal cell lines were obtained by limited dilution method. VASN monoclonal antibodies were prepared in large quantities by ascites-induced biogenesis and purified by using rProtein G. The affinity and specificity of the monoclonal antibodies were detected by ELISA, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), Western blotting, as well as the expression of VASN in tree shrew immortalized fibroblasts and tree shrew tissues. Results The full-length sequence of the tree shrew Vasn gene was successfully amplified and the recombinant plasmid vector of tree shrew pET30a-VASN was constructed. The sequenced sequence of the recombinant plasmid vector was consistent with that of the target gene of tree shrew Vasn. The protein expressed by recombinant vector mainly existed in the form of inclusion bodies. The purity of recombinant VASN protein after purification reached 90%, which met the requirements of subsequent immune experiments. The titer of serum antibody reached 1∶729 000 after four times immunization with recombinant tree shrew VASN protein,and the monoclonal antibody was obtained by ascites induction method and purification, and the constant affinity value of monoclonal antibody was determined by ELISA method to reach 2.59x10⁷mol/L. Western blotting analysis of monoclonal antibody specificity showed that the monoclonal antibody could bind to the recombinant protein of tree shrew VASN, but had no binding reaction with porcine Retinol-Binding Protein 4 (RBP4), human VASN-Leucine Rich Repeat (LRR) and Bovine Serum Albumin (BSA) standard protein. Western blotting detection of VASN expression in major tissues of tree shrew showed that specific bands with clear background were found between anti-VASN monoclonal antibody and major organ tissues of tree shrew (heart, liver, spleen, lung, kidney and muscle) at a relative molecular weight of about 70 000. The results of qRT-PCR showed that the mRNA expression level of Vasn in spleen tissue and lung tissue of tree shrew was relatively high. Immunohistochemical detection using monoclonal antibody of tree shrew VASN showed positive immunohistochemical results in spleen tissue, lung tissue and immortalized fibrocytes of tree shrew with relatively high expression level of Vasn mRNA. However, the tissues with low relative expression level of Vasn mRNA showed negative results. Conclusions In this study, monoclonal antibody against VASN was successfully prepared, which can be used for immunohistochemical detection of immortal fibrocytes, spleen tissue and lung tissue of tree shrew. The study results provide an important tool for further research on the function of VASN in tree shrew models.

Key words: Vasorin (VASN), Tree shrew, Monoclonal antibody

中图分类号:

Q95-33

欧美珍,李泳锋,温莎,等.树鼩VASN单克隆抗体的制备及其应用探索[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2025.027.

OU Meizhen,LI Yongfeng,WEN Sha,et al. Preparation of Monoclonal Antibody to VASN in Tree Shrew and Exploration of Its Application[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2025.027.

图/表 4

图1 Vasn基因克隆与重组注：A， Vasn基因扩增产物；1：Vasn扩增产物B，单克隆菌落PCR鉴定；1~5：Vasn单克隆菌落；C，重组质粒双酶切鉴定；1~2：pET30-VASN双酶切产物；D， Vasn基因测序结果。M：DNA Maker。

Figure 1 Vasn gene cloning and recombinationNote：A， Vasn gene amplification product.1： Vasn amplification product B， PCR identification of monoclonal colonies. 1~5： Vasn monoclonal colonies. C， Recombinant plasmid double digestion identification. 1~2： pET30-VASN double digest product. D， Vasn gene sequencing results. M： DNA Maker.

图2 重组蛋白的表达与纯化注：A，VASN重组蛋白的表达，M：蛋白标志物，1：未诱导pET30a-VASN上清，2：未诱导pET30a-VASN沉淀，3：经诱导pET30a-VASN上清 4：经诱导pET30a-VASN沉淀；B，VASN重组蛋白纯化，M：蛋白标志物，1：纯化前 2：纯化后 3：蛋白上样缓冲液。

Figure 2 Expression and purification of recombinant proteinsNote：A， Expression of VASN recombinant protein. 1： uninduced pET30a-VASN supernatant， 2： uninduced pET30a-VASN precipitate， 3： induced pET30a-VASN supernatant 4： induced pET30a-VASN precipitate. B， VASN recombinant protein purification. M： protein marker， 1： before purification 2： after purification 3： protein loading buffer.

图3 单克隆抗体的制备注：A，小鼠血清效价；B，细胞融合；C，单克隆抗体腹水纯化（M为蛋白标志物，1为纯化TV5-B11）；D，单克隆抗体亲和力测定；E，抗体特异性鉴定（M为蛋白标志物，1为TS-VASN重组蛋白，2为Pig-RBP4重组蛋白，3为LRR重组蛋白，4为BSA标准蛋白）；F，抗体梯度浓度鉴定（M为蛋白标志物，1为0.5 μg，2为1.0 μg，3为1.5 μg，4为2.0 μg）。

Figure 3 Preparation of monoclonal antibodiesNote：A， Mouse serum titer； B， Cell fusion； C， Monoclonal antibody ascites purified （M， protein marker； 1， purified TV5-B11）； D， Monoclonal Antibody Affinity Measurement； E， Antibody specificity identification （M， protein marker； 1， TS-VASN recombinant protein； 2， Pig-RBP4 recombinant protein； 3， LRR recombinant protein； 4， BSA standard protein）； F， Antibody gradient concentration identification （M， Protein marker； 1， 0.5 μg； 2， 1.0 μg； 3， 1.5 μg； 4， 2.0 μg）.

图4 树鼩VASN抗体的应用分析注：A，Western blotting鉴定树鼩各组织中VASN的蛋白表达水平（M为蛋白标志物，qRT-PCR检测树鼩各组织中VASN mRNA相对表达水平）；B，VASN基因扩增（M，DNA Maker；1，阳性对照；2，VASN扩增产物；3，空白对照）；C，免疫组化鉴定细胞中VASN蛋白表达；D，免疫组化鉴定脾脏组织中VASN蛋白表达；E，免疫组化鉴定肺脏组织中VASN蛋白表达。

Figure 4 Application analysis of VASN antibody in tree shrewNote：A， Western blotting identification of VASN protein expression levels in various tissues of tree shrews （M， protein marker； Relative expression levels of VASN mRNA in various tissues of tree shrews detected by qRT-PCR）； B， VASN gene amplification （M， DNA Maker； 1， positive control； 2， VASN amplification product； 3， blank control）； C， Immunohistochemistry to identify VASN protein expression in cells； D， Immunohistochemical identification of VASN protein expression in spleen tissue. E， Immunohistochemical identification of VASN protein expression in lung tissue.

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