WO2022057696A2 - 一种重组蛋白的编码序列、重组蛋白及其单克隆抗体的制备方法 - Google Patents

一种重组蛋白的编码序列、重组蛋白及其单克隆抗体的制备方法 Download PDF

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WO2022057696A2
WO2022057696A2 PCT/CN2021/117134 CN2021117134W WO2022057696A2 WO 2022057696 A2 WO2022057696 A2 WO 2022057696A2 CN 2021117134 W CN2021117134 W CN 2021117134W WO 2022057696 A2 WO2022057696 A2 WO 2022057696A2
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recombinant protein
coding sequence
expression vector
tag
protein expression
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WO2022057696A3 (zh
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李晓照
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中南大学湘雅医院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

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  • the present invention belongs to the field of biotechnology. Specifically, the present invention relates to a coding sequence of a recombinant protein, a method for preparing the recombinant protein and a monoclonal antibody thereof.
  • Tag-antibody is a kind of affinity-purified mouse monoclonal antibody. It is used to detect the tag sequences (such as MyC, His, GST, HA, etc.) on various commercial expression vectors, so as to analyze the expression content and function of the target protein. The principle is antigen-antibody reaction, and these tag antibodies can bind to the corresponding tag fusion protein with high specificity. Tag antibodies are common tools for gene protein expression, signal transduction and gene function studies.
  • the immunogen used in the preparation of conventional monoclonal antibodies to tagged proteins is a complete protein expressed by genetic engineering technology.
  • the protein due to the base codons, the protein is difficult to express in E. coli, and the expression level is extremely low, which leads to subsequent purification work. It is difficult to carry out, which seriously hinders the preparation of its monoclonal antibody.
  • the present invention aims to avoid the deficiencies in the background technology, by designing a recombinant protein and preparing its monoclonal antibody, so as to realize the simultaneous preparation of multiple tag protein monoclonal antibodies, which not only enhances the detection sensitivity, but also can simultaneously prepare a variety of tag protein monoclonal antibodies. antibody.
  • the coding sequence of the recombinant protein is shown in SEQ ID NO.1, specifically:
  • amino acid sequence of the recombinant protein is shown in SEQ ID NO.2, specifically:
  • the recombinant protein expression vector is a plasmid vector containing the coding sequence shown in SEQ ID NO.1.
  • the above recombinant proteins and recombinant protein expression vectors can be used to prepare monoclonal antibodies to tagged proteins.
  • the preparation method of the tag protein monoclonal antibody comprises the following steps:
  • the recombinant protein was immunized to Balb/c mice for many times, and the mouse spleen cells were fused with sp2/0 myeloma cells. After multiple rounds of screening and multi-index identification, monoclonal cells corresponding to the recombinant protein were obtained.
  • the monoclonal antibody to the tag protein was prepared from the monoclonal cell line of the tag protein.
  • the present invention firstly uses four tag-binding proteins (6xHis, S Tag, Trx and GST) as target antigens, and the sequence comparison results show that the selected epitope has no obvious homology with other protein sequences.
  • the selected four dominant epitope sequences were repeated and connected by flexible fragments (four consecutive glycines).
  • the amino acid sequence of the recombinant protein is formed.
  • the amino acid sequence of the recombinant protein is converted into a corresponding nucleotide sequence by using the E. coli preferred codons, so as to facilitate the expression of the recombinant protein in E. coli to increase the expression level.
  • the nucleotide sequence obtained in the previous step is chemically synthesized, and the synthesized nucleotide fragment is inserted into the expression vector PET-28a(+) by enzymatic ligation to construct a recombinant protein expression vector.
  • the recombinant protein expression vector is transformed into Escherichia coli ER2566 competent cells, and the recombinant protein expression strain is obtained by screening.
  • the sixth step after large-scale cultivation of the recombinant protein expression strain, ultrasonic sterilization and low temperature centrifugation, the supernatant of the solution is taken and subjected to affinity chromatography on a nickel agarose affinity chromatography column, and the purified recombinant protein is obtained by elution.
  • the seventh step after immunizing BALB/c mice with the purified recombinant protein for many times, the spleen cells are fused with sp2/0 myeloma cells, and after multiple rounds of screening, hybridoma cell lines are finally obtained respectively.
  • BALB/c mouse ascites was prepared from the hybridoma cell lines respectively, and the monoclonal antibody was purified in two steps using the octanoic acid-ammonium sulfate method and Protein G.
  • the recombinant protein used as an immunogen contains only four tag protein epitopes, which ensures that the final monoclonal antibody only specifically recognizes the four tag proteins, and screening obtains four tag proteins at the same time, which improves the detection sensitivity , reduce the experimental cost.
  • the third is to optimize the nucleotide sequence corresponding to the recombinant protein by using the preferred codons of Escherichia coli, thereby greatly improving the expression level of the recombinant protein in Escherichia coli.
  • the amino acid sequence encoding the recombinant protein is converted into the corresponding nucleotide sequence according to the preferred codons of E. coli.
  • the specific sequence is shown in the sequence table. As shown in SEQ ID NO.1, and the nucleotide sequences corresponding to the enzyme cleavage sites BamHI and EcoRI were added to the upstream and downstream of the nucleotide sequence, it was synthesized by Hangzhou Xianzhi Biotechnology Co., Ltd. The synthesized target gene was cloned into pMD19-T vector (Bao Bioengineering Dalian Co., Ltd.).
  • the pMD19-T vector containing the target gene and the PET-28a(+) vector (Novagen, Germany) were double digested with restriction endonucleases BamHI and EcoRI (Bao Bioengineering Dalian Co., Ltd.) at 37°C for 12 hours.
  • the products were subjected to 1% agarose gel electrophoresis, respectively, and the target gene and PET-28a(+) vector were recovered by cutting the gel (the gel recovery kits used in the present invention were all from Ningbo Zhongding Biotechnology Co., Ltd.).
  • T4 ligase (Bao Bioengineering Dalian Co., Ltd.) to connect the recovered target gene and PET-28a(+) vector at a certain ratio at 4 °C for 12 hours, and then transform the ligation product into DH5 ⁇ competent cells (Hangzhou Xianzhi Biotechnology). Co., Ltd.), and spread it on the LB plate containing kanaicillin resistance (50 ⁇ g/mL), after culturing at 37°C for 12 hours, pick monoclonal strains on the plate to contain kanaicillin resistance (50 ⁇ g/mL).
  • plasmid purification kit (the plasmid purification kits used in the present invention are all from Ningbo Zhongding Biotechnology Co., Ltd.) to extract plasmids, and the plasmids were extracted by BamHI and The correct recombinant expression vector was obtained after EcoRI double digestion and identification.
  • the constructed recombinant expression vector was transformed into E. coli ER2566 competent cells, spread on LB plates containing kanapenicillin resistance (50 ⁇ g/mL), and cultured at 37°C overnight. On the second day, pick the monoclonal strains on the plate to LB liquid medium containing kanapenicillin resistance (50 ⁇ g/mL), cultivate at 37°C for 8 hours on a constant temperature shaker, add the inducer isopropylthio- ⁇ - D-galactoside (final concentration of 1.0 mmol/L) induced expression for 4 hours to prepare protein electrophoresis samples. The results of 13.5% polyacrylamide gel electrophoresis showed that the recombinant protein was successfully expressed, and the recombinant protein expression strain was obtained.
  • the bacteria were collected by centrifugation, the bacteria were disrupted by low-temperature sonication. After low-temperature centrifugation, the supernatant was taken and passed through a nickel agarose affinity chromatography column. After washing and elution, the purified recombinant protein was finally obtained.
  • the two antigenic epitope sequences of tag protein A and B are respectively linked with a cysteine at the N-terminus to synthesize G and H sequence polypeptides.
  • the carrier protein is selected from BSA (Roche Company), and the synthesized peptides are connected by SPDP (PIERCE Company) ligation method.
  • the polypeptides were coupled to BSA: 4.6 mg SPDP dissolved in 740 ul DMSO, the final concentration was 20 mM. 0.1008g of BSA was dissolved in 2ml of PBS-EDTA solution and allowed to stand at room temperature for 1h. HiTrapTM Deaslting column eluted excess SPDP.
  • mice 5-7 weeks old female BALB/c mice were taken, and each mouse was subcutaneously injected with 70 ⁇ g recombinant protein emulsified in Freund's complete adjuvant at multiple points for basic immunization; 15 days later, booster immunization was performed by taking the same amount of recombinant protein with After the incomplete adjuvant was emulsified, it was injected subcutaneously at multiple points; the third booster immunization was 15 days later, and the method was the same as the second time.
  • Proteins BSA-G, BSA-H, I, L were diluted with the coating solution (final concentration was 1 ⁇ g/mL), 100 ⁇ L/well was added to the microtiter plate (Shenzhen Jincanhua Industrial Co., Ltd.), and coated at 4°C for 12 hours Then, wash with washing solution and pat dry; add blocking solution, 150 ⁇ L/well, block at 37°C for 2 hours, discard the liquid in the well, and pat dry; add the cell culture supernatant to be tested and control serum, 100 ⁇ L/well, incubate at 37°C After 1 hour, the washing solution was washed three times and patted dry; HRP (horseradish peroxidase)-labeled goat anti-mouse IgG was added, 100 ⁇ L/well, after incubation at 37°C for 30 minutes, the washing solution was washed four times and patted dry; Add 50 ⁇ L of chromogenic solution A and 50 ⁇ L of chromogenic solution B to
  • Coating solution Na 2 CO 3 1.5 g, NaHCO 3 2.9 g, add double distilled water to dilute to 1000 mL (pH 9.6).
  • Blocking solution Na 2 HPO 4 .12H 2 O 2.68 g, NaH 2 PO 4 .2H 2 O 0.39 g, NaCl 8.5 g, 20 g bovine serum albumin, add double distilled water to make up to 1000 mL (pH 7.4).
  • Washing solution Na 2 HPO 4 .12H 2 O 2.68 g, NaH 2 PO 4 .2H 2 O 0.39 g, NaCl 8.5 g, Tween-20 0.5 mL, add double distilled water to make up to 1000 mL (pH 7.4).
  • Chromogenic Solution A Dissolve 200 mg of TMB in 100 mL of absolute ethanol, and add double distilled water to dilute to 1000 mL.
  • Color developing solution B 2.1 g of citric acid, 71 g of Na 2 HPO 4 .12H 2 O, add double distilled water to dilute to 1000 mL.
  • Stop solution 2M H2SO4 , 21.7mL concentrated H2SO4 and double distilled water to make up to 1000mL.
  • subcloning was performed using limiting dilution. After three subcloning, 6xHis 3 hybridoma cell lines (1F4, 8B6, 3D7, ), 2 S Tag hybridoma cell lines (5D6, 2C3), 5 Trx hybridoma cell lines (4F4, 6B7, 3G7, 6T5, 7H5); GST3 hybridoma cell lines (7F4, 2C7, 6D7).
  • the ascites of BALB/c mice was prepared from the hybridoma cell lines, and the monoclonal antibody was purified in two steps using the octanoic acid-ammonium sulfate method and Protein G.

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Abstract

本发明公开了一种重组蛋白的编码序列、重组蛋白及其单克隆抗体的制备方法。该重组蛋白包含6xHis、S Tag、Trx和GST四种标签蛋白表位，并结合大肠杆菌偏爱密码子将该重组氨基酸序列转换为对应的核苷酸序列，通过分子生物学技术制备重组抗原，免疫小鼠，通过酶联免疫等筛选平台分别获得针对四种标签蛋白的优质单克隆细胞株。

Description

一种重组蛋白的编码序列、重组蛋白及其单克隆抗体的制备方法 技术领域
本发明属于生物技术领域。具体而言，本发明涉及一种重组蛋白的编码序列、重组蛋白及其单克隆抗体的制备方法。
背景技术
标签蛋白抗体(Tag-antibody)是一类经过亲和纯化的小鼠单克隆抗体。用于检测各种商品化表达载体上的标签序列(如：MyC、His、GST、HA等)，籍以分析检验目的蛋白的表达含量及其功能。其原理是抗原-抗体反应，这些标签抗体可以高度特异地结合对应的标签融合蛋白。标签抗体是开展基因蛋白表达、信号转导和基因功能研究的常用工具。
常规的标签蛋白单克隆抗体制备所使用的免疫原是基因工程技术表达的完整蛋白，但由于碱基密码子的缘故，该蛋白在大肠杆菌中表达困难，表达量极低，导致后续的纯化工作难以开展，严重阻碍其单克隆抗体的制备。另外，标签蛋白种类繁多，每种标签蛋白单独制备，耗时长、成本高，不利于市场的广泛推广和使用。
发明内容
本发明旨在避免背景技术中的不足之处，通过设计一种重组蛋白并制备其单克隆抗体，从而实现同时制备多种标签蛋白单克隆抗体，既增强了检测灵敏度，又可以同时制备多种抗体。
为了达到上述目的，本发明提供的技术方案为：
所述重组蛋白的编码序列如SEQ ID NO.1所示，具体为：
Figure PCTCN2021117134-appb-000001
Figure PCTCN2021117134-appb-000002
所述重组蛋白的氨基酸序列如SEQ ID NO.2所示，具体为：
Figure PCTCN2021117134-appb-000003
Figure PCTCN2021117134-appb-000004
所述重组蛋白表达载体是含有SEQ ID NO.1所示编码序列的质粒载体。
上述重组蛋白和重组蛋白表达载体可用于制备标签蛋白单克隆抗体。
所述标签蛋白单克隆抗体的制备方法包括如下步骤：
(1)合成SEQ ID NO.1所示的重组蛋白编码序列，并将该重组蛋白编码序列连接质粒载体，进而构建重组蛋白表达载体；
(2)将重组蛋白表达载体转化大肠杆菌，筛选得到重组蛋白表达菌株；
(3)将重组蛋白表达菌株大规模培养后，经纯化获取重组蛋白，所述重组蛋白的氨基酸序列如SEQ ID NO.2所示；
(4)将重组蛋白多次免疫Balb/c小鼠，取小鼠脾脏细胞与sp2/0骨髓瘤细胞融合，经过多轮筛选和多指标鉴定分别获得针对重组蛋白所对应的标签蛋白单克隆细胞株，再由标签蛋白单克隆细胞株制得标签蛋白单克隆抗体。
下面对本发明作进一步说明：
本发明首先以4种标签结合蛋白(6xHis、S Tag、Trx和GST)为靶抗原，序列比较结果显示所选择的该抗原表位与其它蛋白序列无明显同源 性。
其次，为了促进所选择抗原表位对BALB/c小鼠免疫系统的刺激，增强免疫效果，故分别将所选择的四个优势抗原表位序列重复后通过柔性片段(连续四个甘氨酸)连接，形成重组蛋白氨基酸序列。
第三步，采用大肠杆菌偏爱密码子，将重组蛋白氨基酸序列转换为对应的核苷酸序列，以利于重组蛋白在大肠杆菌中的表达以提高表达量。
第四步，化学合成上一步骤得到的核苷酸序列，并通过酶切连接，将合成得到的核苷酸片段插入表达载体PET-28a(+)，构建重组蛋白表达载体。
第五步，重组蛋白表达载体转化大肠杆菌ER2566感受态细胞，筛选得到重组蛋白表达菌株。
第六步，重组蛋白表达菌株大规模培养后，超声破菌并低温离心后，取溶液上清通过镍琼脂糖亲和层析柱亲和层析，洗脱得到纯化重组蛋白。
第七步，纯化后的重组蛋白多次免疫BALB/c小鼠后，取其脾脏细胞与sp2/0骨髓瘤细胞融合，经过多轮筛选并最终分别得到杂交瘤细胞株。
第八步，将杂交瘤细胞株分别制备BALB/c小鼠腹水，使用辛酸-硫酸铵法及Protein G分两步纯化单克隆抗体。
与现有技术相比，本发明的有益效果为：
一是通过分子生物学技术，实现了四种标签结合蛋白抗原表位的重复及串联表达，增强目的抗原表位对小鼠免疫系统的刺激，排除了无关序列可能带来的干扰；
二是作为免疫原的重组蛋白仅含有四种标签蛋白抗原表位，保证了最终得到的单克隆抗体仅特异性识别该四种标签蛋白，并筛选同时得到四种标签蛋白，提高了检测的灵敏度，降低实验成本。
三是采用大肠杆菌偏爱密码子优化重组蛋白对应的核苷酸序列，从而大大提高了重组蛋白在大肠杆菌中的表达水平。
具体实施方式
一、4种标签蛋白抗原表位选择
以4种标签蛋白为靶抗原，利用生物软件DNAssist2.0分析其抗原表 位序列的亲水性及抗原性，分别选择6xHis、S Tag、Trx和GST抗原表位A、B、C和D。同时，序列比较结果表明所选择的A、B、C和D抗原表位序列特异性高，与其它蛋白序列无明显同源性。
二、4种标签蛋白抗原表位的串联
为增强所选择抗原表位对小鼠免疫系统的刺激以利于后续实验的进行，将标签蛋白A、B、C和D四种抗原表位序列分别通过柔性片段(连续四个甘氨酸)连接，得到重组蛋白氨基酸序列，其具体序列如序列表SEQ ID N0.2所示。
三、优化编码重组蛋白的核苷酸序列
为了提高重组蛋白在大肠杆菌中的表达量，在重组蛋白氨基酸序列不变的前提下，根据大肠杆菌偏爱密码子将编码重组蛋白的氨基酸序列转化为对应的核苷酸序列，具体序列如序列表SEQ ID NO.1所示，并在其上下游分别添加酶切位点BamHI和EcoRI对应的核苷酸序列后，由杭州贤至生物科技有限公司合成。合成后的目的基因克隆于pMD19-T载体(宝生物工程大连有限公司)中。
四、构建重组蛋白表达载体
用限制性内切酶BamHI和EcoRI(宝生物工程大连有限公司)于37℃分别双酶切含目的基因的pMD19-T载体和PET-28a(+)载体(德国Novagen公司)12小时，酶切产物分别行1％琼脂糖凝胶电泳，并分别切胶回收目的基因和PET-28a(+)载体(本发明所使用的胶回收试剂盒均来自宁波中鼎生物技术有限公司)。使用T4连接酶(宝生物工程大连有限公司)将回收的目的基因和PET-28a(+)载体按一定的比例于4℃连接12小时后，连接产物转化DH5α感受态细胞(杭州贤至生物科技有限公司)，并涂布于含卡那青霉素抗性(50μg/mL)的LB平板，于37℃恒温培养12小时之后，于平板上挑取单克隆菌株至含卡那青霉素抗性(50μg/mL)的LB液体培养基，37℃恒温摇床培养12小时后，采用质粒纯化试剂盒(本发明所使用的质粒纯化试剂盒均来自于宁波中鼎生物技术有限公司)提取质粒，经BamHI和EcoRI双酶切鉴定后得到正确的重组表达载体。
五、构建重组蛋白E表达菌株
将构建好的重组表达载体转化E.coli ER2566感受态细胞，并涂布于含卡那青霉素抗性(50μg/mL)的LB平板，于37℃过夜培养。第二日，挑取平板上单克隆菌株至含卡那青霉素抗性(50μg/mL)的LB液体培养基，37℃恒温摇床培养8小时后，加诱导剂异丙基硫代-β-D-半乳糖苷(终浓度为1.0mmol/L)诱导表达4个小时后制备蛋白电泳样品。13.5％聚丙烯酰胺凝胶电泳结果表明重组蛋白成功表达，得到重组蛋白表达菌株。
六、纯化重组蛋白F
接种重组蛋白表达菌株至LB液体培养基，加卡那青霉素至终浓度为50μg/mL，37℃恒温摇床培养8小时后，用含50μg/mL卡那青霉素的LB液体培养基将该菌按1：100比例稀释后，分装至细菌培养瓶中，置37℃恒温摇床培养至OD600＝0.8，加诱导剂异丙基硫代-β-D-半乳糖苷至终浓度为1.0mmol/L，继续培养诱导4小时。离心收集菌体后，低温超声破菌，低温离心后取上清通过镍琼脂糖亲和层析柱，经洗涤、洗脱最终得到纯化重组蛋白。
七、构建用于检测的偶联蛋白
将标签蛋白A、B两个抗原表位序列分别在N端连接一个半胱氨酸，合成G、H序列多肽，载体蛋白选择BSA(Roche公司)，用SPDP(PIERCE公司)连接法将合成的多肽分别与BSA偶联：4.6mg SPDP溶解740ul DMSO，终浓度为20mM。0.1008g BSA溶解于2ml PBS-EDTA溶液中，室温静置1h。HiTrapTM Deaslting column脱盐柱洗脱多余的SPDP。4mg多肽加入偶联好的BSA-SPDP体系中室温过夜，得到产物BSA-G、BSA-H(由杭州贤至生物科技有限公司合成)。将蛋白GST和TRX(购买于杭州毕肯莱博生物科技有限公司)命名为I、L。
八、杂交瘤细胞株的获得
取5-7周龄雌性BALB/c小鼠，基础免疫每只小鼠皮下多点注射福氏完全佐剂乳化的70μg重组蛋白；15天后进行加强免疫，方法为取相同量的重组蛋白用福氏不完全佐剂乳化后，皮下多点注射；第三次加强免疫在 15天以后，方法同第二次相同。30天后，取120μg重组蛋白腹腔加强注射，并于腹腔加强注射72小时后，眼眶取血，并处死小鼠，取其脾脏制备细胞悬液，细胞计数，按1/5于脾细胞的数量取生长状态良好的sp2/0小鼠骨髓瘤细胞，混和离心后，加入聚乙二醇(PEG-4000)使二者融合。另外，再加入等体积的饲养细胞，混匀后分置于96孔细胞板(200μL/孔)，于5％二氧化碳培养箱培养。5天后，半保留换液，10天后采用间接酶联免疫吸附法检测96孔细胞培养板中的杂交瘤细胞培养上清。
具体方法如下：
蛋白BSA-G、BSA-H、I、L分别通过包被液稀释后(终浓度为1μg/mL)，以100μL/孔加入酶标板(深圳金灿华实业有限公司)，4℃包被12小时后用洗涤液洗涤一次并拍干；加入封闭液，150μL/孔，37℃封闭2小时，弃孔内液体，拍干；分别加待检细胞培养上清及对照血清，100μL/孔，37℃孵育1小时后，洗涤液洗涤三次并拍干；加HRP(辣根过氧化物酶)标记的羊抗鼠IgG，100μL/孔，37℃孵育30分钟后，洗涤液洗涤四次并拍干；每孔加显色液A和显色液B各50μL，37℃避光显色10分钟后，加终止液终止反应，50μL/孔，酶标仪450nm波长空白孔校零后读取OD值。以免疫小鼠的血清作为阳性对照，相关溶液配方如下：
包被液：Na 2CO 31.5g，NaHCO 32.9g，加双蒸水定容至1000mL(pH9.6)。
封闭液：Na 2HPO 4.12H 2O 2.68g，NaH 2PO 4.2H 2O 0.39g，NaCl 8.5g，20g牛血清白蛋白，加双蒸水定容至1000mL(pH7.4)。
洗涤液：Na 2HPO 4.12H 2O 2.68g，NaH 2PO 4.2H 2O 0.39g，NaCl 8.5g，Tween-20 0.5mL，加双蒸水定容至1000mL(pH7.4)。
显色液A:200mg TMB溶于100mL无水乙醇，加双蒸水定容至1000mL。
显色液B:柠檬酸2.1g，Na 2HPO 4.12H 2O 71g，加双蒸水定容至1000mL。
使用时：1mL显色液A+1mL显色液B+0.4μL 30％H 2O 2。
终止液：2M H 2SO 4，21.7mL浓H2SO4加双蒸水定容至1000mL。
对于检测阳性的杂交瘤细胞克隆，再使用有限稀释法进行亚克隆。经 过三次亚克隆，分别筛选得到6xHis 3株杂交瘤细胞株(1F4、8B6、3D7、)，S Tag 2株杂交瘤细胞株(5D6、2C3)；Trx 5株杂交瘤细胞株(4F4、6B7、3G7、6T5、7H5)；GST3株杂交瘤细胞株(7F4、2C7、6D7)。
最后将杂交瘤细胞株分别制备BALB/c小鼠腹水，使用辛酸-硫酸铵法及Protein G分两步纯化单克隆抗体。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (7)

  1. 一种重组蛋白的编码序列，其特征在于，所述编码序列如SEQ ID NO.1所示。
  2. 如权利要求1所述编码序列编码的重组蛋白，其特征在于，所述重组蛋白的氨基酸序列如SEQ ID NO.2所示。
  3. 一种重组蛋白表达载体，其特征在于，所述重组蛋白表达载体是含有SEQ ID NO.1所示编码序列的质粒载体。
  4. 根据权利要求3所述的重组蛋白表达载体，其特征在于，所述重组蛋白表达载体的原始质粒为PET-28a(+)。
  5. 如权利要求2所述重组蛋白在制备标签蛋白单克隆抗体中的应用。
  6. 如权利要求3或4所述重组蛋白表达载体在制备标签蛋白单克隆抗体中的应用。
  7. 一种标签蛋白单克隆抗体的制备方法，其特征在于，所述方法包括如下步骤：
    (1)合成SEQ ID NO.1所示的重组蛋白编码序列，并将该重组蛋白编码序列连接质粒载体，进而构建重组蛋白表达载体；
    (2)将重组蛋白表达载体转化大肠杆菌，筛选得到重组蛋白表达菌株；
    (3)将重组蛋白表达菌株大规模培养后，经纯化获取重组蛋白，所述重组蛋白的氨基酸序列如SEQ ID NO.2所示；
    (4)将重组蛋白多次免疫Balb/c小鼠，取小鼠脾脏细胞与sp2/0骨髓瘤细胞融合，经过多轮筛选和多指标鉴定分别获得针对重组蛋白所对应的标签蛋白单克隆细胞株，再由标签蛋白单克隆细胞株制得标签蛋白单克隆抗体。
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