WO2018068244A1 - 一种单链抗体及其在特异性检测正平行型g-四链体中的应用 - Google Patents

一种单链抗体及其在特异性检测正平行型g-四链体中的应用 Download PDF

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WO2018068244A1
WO2018068244A1 PCT/CN2016/101945 CN2016101945W WO2018068244A1 WO 2018068244 A1 WO2018068244 A1 WO 2018068244A1 CN 2016101945 W CN2016101945 W CN 2016101945W WO 2018068244 A1 WO2018068244 A1 WO 2018068244A1
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quadruplex
chain antibody
parallel
positive
dna
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French (fr)
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黄志纾
刘慧云
殴田苗
赵勇
古练权
谭嘉恒
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中山大学
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

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  • the present invention belongs to the field of biological detection technology, and more particularly to a single-chain antibody and its use in specifically detecting a positive parallel type G-quadruplex.
  • telomere G-quadruplex The conformational confirmation of telomere G-quadruplex has always been a hotspot in the field of G-quadruplex research. It has been shown that telomere G-quadruplexes form different conformations in different solution environments, for example, In K + solution, the telomere-rich G sequence forms a mixed G-quadruplex. In Na + solution, the telomere-rich G sequence forms an anti-parallel G-quadruplex, while in Sr 2+ solution, telomere The G-rich sequence will form a positive parallel G-quadruplex. In addition, molecular crowding, high temperature and other solution environments also promote the formation of telomere positive parallel G-quadruplex. However, these findings are only limited to the in vitro solution environment, and there is still no evidence for the conformational confirmation of the telomere-rich G sequence in the intracellular environment.
  • G-quadruplex a special nucleic acid secondary structure involved in life events and whether it can become a drug target has also attracted wide attention of researchers. Therefore, whether G-quadruplex can stably exist in vivo and whether G-quadruplex small molecule ligand can directly target G-quadruplex DNA becomes a key problem.
  • G-quadruplex antibodies have developed into useful tools for studying intracellular G-quadruplexes.
  • Christiane Schaffitzel et al. used the ribosome display technology to screen the G-quadruplex antibody Sty49, and used this antibody to detect the telomere G-quadruplex DNA in the prokaryotic echinococcosis.
  • the object of the present invention is to provide a single chain antibody according to the deficiencies in the prior art.
  • Another object of the present invention is to provide the use of the above single-chain antibody for specifically detecting a positive parallel type G-quadruplex.
  • the present invention provides a single-chain antibody, and the gene sequence encoding the single-chain antibody is shown in SEQ ID NO: 1.
  • the single-chain antibody designed by the invention can selectively recognize the positive parallel conformation G-quadruplex DNA in the in vitro enzyme-linked immunosorbent assay, and the mixed G-quadruplex and anti-parallel G-quadruplex DNA detection Not combined.
  • the purified antibody was used for the detection of intracellular G-quadruplex, and it was also found that this antibody selectively recognizes a positive parallel conformation G-quadruplex. Therefore, the single-chain antibody fragment of the present invention can be used for the study of endogenous G-quadruplex DNA of cells.
  • genomic DNA and telomeric DNA as an example to illustrate the use of immunofluorescence and chromatin immunoprecipitation to detect the secondary structure of a G-quadruplex nucleic acid in a positive parallel conformation is illustrated.
  • the present invention also provides a method for preparing a single-chain antibody, which inserts the gene sequence shown in SEQ ID NO: 1 into a prokaryotic expression vector to obtain a recombinant plasmid, and then transforms the recombinant plasmid into E. coli for expression and purification.
  • the present invention also provides the use of the above single-chain antibody for specifically detecting a positive parallel type G-quadruplex.
  • the above-described single-chain antibody can be applied to prepare a material for specifically detecting a positively parallel type G-quadruplex, for example, to prepare a corresponding kit or test strip material.
  • a method for specifically detecting a positive parallel type G-quadruplex specifically using a single-chain antibody encoded by the gene sequence shown in SEQ ID NO: 1 as a probe for specifically recognizing a positive parallel type G-four-strand body.
  • the immunization method is an ELISA method.
  • an ELISA method for specifically detecting a positive parallel type G-quadruplex the specific step is: binding a biotin-labeled nucleic acid sample to be tested to a streptavidin-coated 96-well plate at 4 ° C Overnight, wash the plate with ELISA buffer, then incubate with blocking solution at room temperature, wash the plate with ELISA buffer after incubation; Enter the single-chain antibody, wash the plate with ELISA buffer after incubation, add HRP-Protein A diluted with blocking solution, and wash the plate with buffer after incubation; then add TMB substrate, develop color, add stop solution to stop the reaction, use The absorbance at 450 nm was measured by a microplate reader.
  • the present invention has the following advantages and beneficial effects:
  • the antibody gene provided by the invention has a short sequence length, simple cloning and convenient expression; the transcribed and translated single-chain antibody fragment (scFv) can specifically detect the G-quadruplex structure recognizing the positive parallel conformation, and realize the positive parallel conformation
  • the G-quadruplex structure is distinguished from other secondary structures, and the positive parallel type G-quadruplex DNA can be detected in vitro by a simple enzyme-linked immunosorbent assay; the antibody protein edited by the gene sequence provided by the present invention is useful as a useful tool.
  • the detection of the positive parallel conformation G-quadruplex in the genome of the cell is realized, and the formation of the G-quadruplex structure of the positive parallel conformation and its corresponding biological function can be detected by a colocalization method at a specific site within the cell.
  • Figure 1 shows the results of purification of the single-chain antibody fragment encoded by the gene sequence, wherein E-1, E-2, E-3, and E-4 are four repeated detections of the single-chain antibody fragment of interest eluted in the purification process, respectively.
  • Figure 2 is a ELISA result of selective binding of a single-chain antibody fragment to a positive-parallel G-quadruplex DNA.
  • Figure 3 is a plot of competitive ELISA for single-chain antibody fragments selectively binding to positive-parallel G-quadruplex DNA.
  • Figure 4 shows the results of immunofluorescence of a single-chain antibody fragment selectively binding to a positive-parallel G-quadruplex DNA at the cellular level.
  • Figure 5 is a measurement of the binding constant of a single-chain antibody fragment to a positive-parallel G-quadruplex DNA (MYC).
  • Figure 6 is a distribution of binding sites for single-chain antibody fragments selectively within the genome.
  • Figure 7 is a G-quadruplex conformational analysis of the binding sequence of a single-chain antibody fragment selectively within the genome.
  • Figure 8 is a bioinformatics analysis of the binding of a single-chain antibody fragment to a telomere positive-parallel G-quadruplex in a living cell.
  • Figure 9 is an analysis of the results of immunoblotting of a single-chain antibody fragment in a living cell in which a telomere positive-parallel G-quadruplex is bound.
  • Figure 10 is a G-quadruplex conformation assay of telomeric DNA in different ionic environments.
  • Figure 11 shows the binding of single-chain antibody fragments to different conformational telomere G-quadruplex DNA binding constants in different ionic environments.
  • Figure 12 shows the results of immunofluorescence of telomere positive-parallel G-quadruplex DNA by co-localization of single-chain antibody fragments with TRF2.
  • Figure 13 is a result of immunofluorescence of a single-chain antibody fragment or colocalization of BG4 with TRF2 to detect the dominant conformation of telomere G-quadruplex.
  • the positive-parallel G-quadruplex antibodies of the invention are screened using phage display technology.
  • the screening antigen was a biotin-labeled DNA sequence capable of forming a positive parallel type G-quadruplex, which was dissolved in 50 mM Tris-HCl (pH 7.4), heated at 95 ° C for 10 minutes, and slowly annealed to room temperature to form a positive Parallel G-quadruplex structure.
  • the phage antibody library was Tomlinson (I+J), purchased from the United Kingdom (Source Bioscience), and the antibody library for positive-parallel G-quadruplex antibody screening was Tomlinson J.
  • the antibody screening process was carried out in strict accordance with the method described by MRC (http://www.geneservice.co.uk/products/proteomic/datasheets/tomlinsonIJ.pdf), but due to the specificity of the G-quadruplex antigen, in the experiment ELISA buffer (50 mM potassium phosphate buffer, pH 7.4, 100 mM potassium chloride) was used instead of PBS to maintain the stability of its positive parallel G-quadruplex conformation, replacing it with streptavidin-labeled magnetic beads.
  • the immune tube is used to separate the antigen and antibody. The simple steps are as follows:
  • biotin-labeled positive-parallel G-quadruplex DNA was coated on streptavidin-labeled magnetic beads and incubated for 15 minutes at room temperature with gentle shaking to wash away unbound DNA and block the surface of the beads with biotin.
  • 2 ⁇ 10 14 antibody-bearing phages were equilibrated with competitive double-stranded DNA for 1 hour, and then added to magnetic beads labeled with a positive parallel type G-quadruplex, and incubated for 1 hour at room temperature to wash away unbound phage.
  • the phage was bound to the positive parallel type G-quadruplex using trypsin digestion.
  • the eluted phage was infected with TG1 E. coli for expansion culture.
  • the obtained phage was infected with HB2151, and single colonies were obtained by plate culture, and about 100 single colonies were picked for expression of single-chain antibodies, and positive clones were verified by ELISA.
  • a single-chain antibody fragment capable of specifically recognizing the positive-parallel G-quadruplex was finally obtained, and its gene sequence was sequenced.
  • the sequence SEQ ID NO: 1 was obtained.
  • nucleic acid samples were purchased from Yingjun Biotechnology Co., Ltd., and the appropriate amount of nucleic acid was dissolved in a buffer of potassium phosphate (ELISA buffer: pH 7.4, 50 mM potassium dihydrogen phosphate, 100 mM KCl), super The concentration was measured by micro-ultraviolet measurement, and then diluted with 5 ⁇ M DNA solution in ELISA buffer, heated at 95 ° C for 5 min, then slowly cooled and annealed to room temperature, and the DNA secondary structure and G-quadruplex conformation of the nucleic acid were determined by circular dichroism.
  • ELISA buffer pH 7.4, 50 mM potassium dihydrogen phosphate, 100 mM KCl
  • GGGGAGGGTGGGGAGGGTGGGGAAGG/CCTTCCCCACCCTCCCCACCCTCCCCA is a duplex sequence
  • the duplex sequence is a double-stranded sequence
  • GGGGAGGGTGGGGAGGGTGGGGAAGG is named duplex-1
  • the sequence number is SEQ ID NO: 13
  • CCTTCCCCACCCTCCCCACCCTCCCCA is named duplex-2
  • the sequence number is SEQ ID NO: 14.
  • the gene sequence shown in SEQ ID NO: 1 was inserted into the prokaryotic expression vector pSANG10 by genetic engineering technology to obtain a recombinant plasmid, and the sequence was confirmed by sequencing.
  • the recombinant plasmid was transformed into E.
  • coli (BL21DE3) for expression and purification: the strain stored at -80 ° C was inoculated into 5 mL of 2 ⁇ TY + 2% glucose + 50 ng / ⁇ l kanamycin in the medium, and placed Incubate in a shaker at 30 ° C, 200 rpm overnight; take 3 mL of the overnight culture inoculated into 300 mL of 2 ⁇ TY + 0.1% glucose + 50 ng / ⁇ l kanamycin medium, put into a shaker at 37 ° C, 250 rpm Incubate for 3 hours.
  • IPTG was added to the medium to a final concentration of 0.2 mM, and placed in a shaker at 25 ° C, incubated at 280 rpm overnight. The overnight cultured bacteria were collected, centrifuged at 4000 rpm for 30 minutes at 4 ° C, and the supernatant was discarded to obtain a bacterial precipitate. The pellet was resuspended in 12 mL of TES and placed on ice for 10 minutes. 18 mL of 5-fold diluted TES (50 mM Tris-HCl, 1 mM EDTA, 20% sucrose, pH 8.0) was added and placed on ice for 15 minutes. Centrifuge at 8000 rpm for 10 minutes at 4 ° C and take the supernatant.
  • 5-fold diluted TES 50 mM Tris-HCl, 1 mM EDTA, 20% sucrose, pH 8.0
  • the supernatant was purified using a nickel column, and after loading, the heteroprotein was washed with 20 mL of a washing solution (PBS + 100 mM NaCl + 10 mM imidazole, pH 8.0). After the protein was rinsed completely, the antibody was eluted with an eluent (PBS + 250 mM imidazole, pH 8.0). The eluted sample was desalted and concentrated by ultrafiltration through an ultrafiltration tube. The concentrated sample was assayed for antibody concentration by BCA and stored in liquid nitrogen at -80 °C.
  • the samples expressing the various stages in the purification process were subjected to SDS-PAGE analysis, and the results are shown in Fig. 1.
  • the eluted sample is the target antibody fragment and has high purity.
  • the single-chain antibody fragments obtained by purification can be used for subsequent activity characterization.
  • ELISA enzyme-linked immunosorbent assay
  • HRP-Protein A diluted 1:5000 with blocking solution was added, and after incubation for 1 hour, the plate was washed 3 times with ELISA buffer containing 0.1% Tween20.
  • the absorbance at 450 nm represents the binding strength of the antibody to DNA, and the higher the absorbance value, the stronger the binding.
  • ELISA enzyme-linked immunosorbent assay
  • the competitive ELISA further validates the specificity of the antibody binding to the positive-parallel G-quadruplex, i.e., the antibody was incubated with an equal amount of competitive DNA for an additional hour prior to the step of adding the antibody by the ELISA assay described above.
  • the results show (Fig.
  • the unbound antibody was washed away with the blocking solution, and the fluorescently labeled secondary antibody and DAPI (nucleated nuclei) were incubated at room temperature for 1 hour at room temperature.
  • the unbound antibody and nuclear dye were washed away with blocking solution, and then confocal microscopy was used. Perform imaging analysis.
  • the binding constant of scFv to MYC, TBA and hTELO was determined by ELISA, and the results were shown (Fig. 5).
  • the binding constant of D1 to the positive parallel conformation G-quadruplex MYC was 27.8 ⁇ 0.9 nM, while the binding of the antiparallel G-quadruplex TBA and the mixed G-quadruplex hTELO was not detected.
  • This result also confirmed that the single-chain antibody fragment edited by the gene sequence of the present invention specifically binds to the positive-parallel conformation G-quadruplex and has high binding affinity.
  • the gene fragment shown in SEQ ID NO: 1 was inserted into the eukaryotic expression vector pEGFP-N3 plasmid and transiently transfected. Single-chain antibody fragments were expressed in live cells for 24 hours. SiHa cells expressing the single-chain antibody fragment were collected, cross-linked with 1% paraformaldehyde for 15 minutes, the cross-linked lysed cells were terminated, and the chromatin was cleaved with Micrococcal Nuclease (Chip Grade) to obtain a chromatin fragment of 200 to 500 bp.
  • Chip Grade Micrococcal Nuclease
  • the chromatin fragment was removed to 10% as an input, and the remainder was subjected to subsequent chromatin immunoprecipitation experiments.
  • the chromatin-positive parallel-type G-quadruplex DNA and the scFv complex were separated with Protein A to finally obtain a scFv-bound DNA fragment derived from the human genome.
  • the DNA fragment obtained in the above experiment was subjected to Hiseq 2500 sequencing analysis, and the results showed (FIG. 6) that the single-chain antibody fragment of the present invention enriched 8345 significant peaks in living cells compared to Input, showing a positive parallel type G-four.
  • the chains are widely distributed in the genome.
  • the binding sites of the single-chain antibody fragments were mainly distributed in the gene region (69.7%), and the rest were distributed in the intergenic region (30.3%).
  • the distribution of the positive parallel G-quadruplex in the gene region is the untranslated region UTR (5%), the promoter transcription start site promoter-TSS (16.8%), and the transcription termination site TTS (1.7%). Proton (14.7%) and intron (30.3%).
  • the oligonucleotide is selected from the first 99 peaks enriched by scFv. Glycosylates were verified by circular dichroism (CD). The results showed (Fig. 7) that most of the DNA fragments formed a G-quadruplex in a positive parallel conformation (91/99, 92%), and the remaining DNA formed a mixed G-quadruplex, while mixing
  • the CD map of the G-quadruplex also has a positive parallel conformational absorption peak as the main peak.
  • the single-chain antibody fragment edited by the gene sequence of the present invention selectively binds to the characteristics of the positive-parallel G-quadruplex in the genome.
  • telomere peaks In order to investigate whether the positive parallel G-quadruplex structure is stably formed in the telomere region of cells, according to the above ChIP-seq data, we Peaks with more than 3 TTAGGG/CCCTAA repeats were set as telomere peaks for further bioinformatics data analysis. The results showed (Fig. 8) that in the scFv ChIP sample, the telomere peak we set was 4.8 times that of the Input sample, indicating that the intracellular telomere positive parallel G-quadruplex was detected by scFv and enriched. .
  • telomere dot blotting analysis Fig. 9
  • the scFv can significantly enrich the telomere sequence, indicating that D1 binds to the positive parallel telomere G-quadruplex in living cells, and also explains The formation of this secondary structure in living cells is stable and exists.
  • telomere-rich G sequence forms G-quadruplex structures with different conformations in different solution backgrounds.
  • the telomere-rich G sequence is annealed in Na + ion solution to form an anti-parallel G-quadruplex, which is annealed in K + ion solution to form a mixed G-quadruplex in Sr 2+ .
  • a positive parallel type G-quadruplex is formed (Fig. 10).
  • scFv single-chain antibody designed by the present invention to explore the main existential conformation of telomere G-quadruplex in cells, and quantitatively studied using two G4 antibodies, BG4 and scFv.
  • the antibody BG4 is capable of binding G-quadruplex DNA of different conformations
  • the scFv edited by the gene sequence of the present invention selectively binds to the G4 DNA of the positive parallel conformation, and the colocalization amount of D1, BG4 and telomere binding protein TRF2 is detected by comparison.
  • telomere G-quadruplex detected by BG4 is also greater than that of the positive parallel G-quadruplex detected by scFv Amount, but the increase is not large (1.3 times), indicating that in addition to the positive parallel conformation G-quadruplex, there are other two conformational G-quadruplexes (mixed and anti-parallel).
  • the positive parallel conformation may be the predominant mode of telomere G-quadruplex in cells (77%).
  • the single-chain antibody provided by the present invention is an excellent macromolecular probe which can be used for structural studies of intra-regional genomes and positive-parallel G-quadruplex DNA of specific functional regions.

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Abstract

提供了一种单链抗体及其在特异性检测正平行型G-四链体中的应用。编码该单链抗体的基因序列如SEQ ID NO：1所示，其转录翻译的单链抗体片段可以特异性地识别正平行构象的G-四链体结构，实现了正平行构象G-四链体结构与其他二级结构的区分，通过酶联免疫吸附试验在体外就能检测正平行型G-四链体DNA。

Description

一种单链抗体及其在特异性检测正平行型G-四链体中的应用 技术领域
本发明属于生物检测技术领域，更具体地，涉及一种单链抗体及其在特异性检测正平行型G-四链体中的应用。
背景技术
端粒G-四链体的构象确证一直以来都是G-四链体研究领域的热点，已有结果表明端粒G-四链体在不同的溶液环境中会形成不同的构象，例如，在K+溶液中，端粒富G序列形成混合型G-四链体，在Na+溶液中，端粒富G序列形成反平行型G-四链体，而在Sr2+溶液中，端粒富G序列则会形成正平行型G-四链体。除此之外，分子拥挤，高温等溶液环境也会促进端粒正平行型G-四链体的形成。但是，这些研究结果都只局限于在体外溶液环境中测得，对于端粒富G序列在细胞内环境下的构象确证依然缺乏相关证据。
G-四链体这一特殊的核酸二级结构参与的生命事件及其是否能成为药物靶点也引起了研究者们的广泛关注。因此，G-四链体在体内是否能够稳定存在以及G-四链体小分子配体能否直接靶向G-四链体结构DNA成为了关键问题。近年来，G-四链体抗体发展成为了研究细胞内G-四链体的有用工具。2001年，Christiane Schaffitzel等利用核糖体展示技术筛选得到的G-四链体抗体Sty49，并利用此抗体检测到了原核生物棘尾虫中端粒G-四链体结构DNA。此研究成果首次证明了体内G-四链体结构的存在。而分别在2013年和2014年，Shankar Balasubramanian等利用噬菌体展示技术筛选得到的高亲和力高选择性G-四链体抗体BG4，首次证明了在哺乳动物细胞中存在DNA G-四链体结构以及RNA G-四链体结构，为体内G-四链体的研究取得了突破性的进展。同时，这些研究结果也表明G-四链体抗体成为了体内G4结构与功能的重要研究工具。目前为止，已发表的用于G4结构与功能研究的G-四链体抗体主要有五个，分别为mevIIB4，Sty49，HF2，BG4,1H6。这些抗体都能够特异性识别G-四链体结构DNA，而与其他的DNA二级结构不结合，但是，它们对不同构象G-四链体的选择性较差，这也限制了这些抗体用于特定功能位点的 G-四链体的结构研究。
发明内容
本发明的目的在于根据现有技术中的不足，提供了一种单链抗体。
本发明的另一目的在于提供上述单链抗体在特异性检测正平行型G-四链体中的应用。
本发明的目的通过以下技术方案实现：
本发明提供了一种单链抗体，编码该单链抗体的基因序列如SEQ ID NO：1所示。
本发明设计的单链抗体在体外酶联免疫吸附试验中可选择性识别正平行型构象G-四链体DNA，而与混合型G-四链体以及反平行型G-四链体DNA检测不到结合。同时，将纯化的抗体用于细胞内G-四链体的检测，也发现此抗体可选择性识别正平行型构象G-四链体。因此，本发明的单链抗体片段可以用于细胞内源性G-四链体DNA的研究。以基因组DNA以及端粒DNA为例来说明本发明的单链抗体片段使用免疫荧光以及染色质免疫沉淀等方法检测正平行构象G-四链体核酸二级结构的应用。
本发明同时提供了一种单链抗体的制备方法，将SEQ ID NO：1所示的基因序列插入到原核表达载体中，获得重组质粒，再将重组质粒转化到大肠杆菌中进行表达与纯化。
本发明同时提供上述单链抗体在特异性检测正平行型G-四链体中的应用。
更近一步地，可以将上述单链抗体应用于制备特异性检测正平行型G-四链体的材料，例如，制备成相应的试剂盒或试纸材料。
一种特异性检测正平行型G-四链体的免疫方法，具体为使用SEQ ID NO：1所示基因序列编码的单链抗体作为探针，用于特异性识别正平行型G-四链体。
优选地，所述的免疫方法为ELISA方法。另外，采用现有其他免疫分析方法特异性检测正平行型G-四链体均在本发明的保护范围之内。
更优选地，一种特异性检测正平行型G-四链体的ELISA方法，具体步骤为：将生物素标记的待测核酸样品结合到链霉亲和素包被的96孔板，4℃过夜，采用ELISA缓冲液洗板，然后采用封闭液室温孵育，孵育后用ELISA缓冲液洗板；加 入单链抗体，孵育后用ELISA缓冲液洗板，加入用封闭液稀释的HRP-Protein A，孵育后，用缓冲液洗板；然后加入TMB底物，显色，加入终止液终止反应，用酶标仪测定450nm的吸光值。
与现有技术相比，本发明具有以下优点及有益效果：
本发明提供的抗体基因序列长度较短，克隆简单，便于表达；其转录翻译的单链抗体片段(scFv)可以特异性地检测识别正平行构象的G-四链体结构，实现了正平行构象G-四链体结构与其他二级结构的区分，用简单的酶联免疫吸附试验可在体外检测正平行型G-四链体DNA；本发明提供的基因序列所编辑的抗体蛋白作为有用工具实现在细胞内基因组中正平行型构象G-四链体的探测，并可通过共定位的方法实现在细胞内特定位点检测正平行型构象G-四链体结构的形成及其相应生物功能。
附图说明
图1为基因序列编码的单链抗体片段纯化结果，其中，E-1、E-2、E-3、E-4分别为纯化过程中洗脱下来的目的单链抗体片段四次重复检测。
图2为单链抗体片段选择性结合正平行型G-四链体DNA的ELISA结果。
图3为单链抗体片段选择性结合正平行型G-四链体DNA的竞争性ELISA结果。
图4为单链抗体片段在细胞水平上选择性结合正平行型G-四链体DNA的免疫荧光结果。
图5为单链抗体片段与正平行型G-四链体DNA(MYC)的结合常数测定。
图6为单链抗体片段选择性在基因组内的结合位点分布。
图7为单链抗体片段选择性在基因组内的结合序列G-四链体构象分析。
图8为单链抗体片段在活细胞内结合端粒正平行型G-四链体的生物信息学结果分析。
图9为单链抗体片段在活细胞内结合端粒正平行型G-四链体的免疫印迹结果分析。
图10为端粒DNA在不同离子环境下的G-四链体构象测定。
图11为单链抗体片段结合在不同离子环境下的不同构象端粒G-四链体DNA结合常数测定。
图12为单链抗体片段与TRF2共定位检测端粒正平行型G-四链体DNA的免疫荧光结果。
图13为单链抗体片段或BG4与TRF2共定位检测端粒G-四链体优势构象的免疫荧光结果。
具体实施方式
以下结合具体实施例和附图来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，本发明所用试剂和材料均为市购。
实施例1
抗体的筛选：本发明的正平行型G-四链体抗体是采用噬菌体展示技术进行筛选的。筛选抗原为生物素标记的能形成正平行型G-四链体的DNA序列，将其溶解于50mM Tris-HCl(pH7.4)，95℃加热10分钟并缓慢退火至室温，使其形成正平行型G-四链体结构。噬菌体抗体库为Tomlinson(I+J)，购买自英国(Source Bioscience)，用于正平行型G-四链体抗体筛选的抗体库为Tomlinson J。抗体筛选过程都严格按照MRC描述的方法开展(http://www.geneservice.co.uk/products/proteomic/datasheets/tomlinsonIJ.pdf)，但由于G-四链体抗原的特殊性，在实验中使用ELISA缓冲液(50mM磷酸钾缓冲液，pH7.4，100mM氯化钾)替代了PBS来维持其正平行型G-四链体构象的稳定，用链霉亲和素标记的磁珠替代了免疫管来实现抗原抗体的分离。简单步骤如下所述：
首先，将生物素标记的正平行型G-四链体DNA包被在链霉亲和素标记的磁珠上，室温轻摇孵育15分钟，洗去未结合的DNA并用生物素封闭磁珠表面。将2×1014个表面携带抗体的噬菌体与竞争性双链DNA平衡1小时之后加入标有正平行型G-四链体的磁珠中，室温孵育1小时，将未结合的噬菌体洗去，与正平行型G-四链体结合噬菌体使用胰酶消化洗脱。洗脱的噬菌体感染TG1大肠杆菌进行扩大培养。历经三轮重复筛选之后，将获得的噬菌体感染HB2151，平板培养获得单菌落，挑取约100个单菌落进行单链抗体的表达，并使用ELISA的方法进行阳性克隆的验证。通过对不同DNA二级结构结合的验证以及对不同构象G-四链体的结合表征，最终 获得能特异性识别正平行型G-四链体的单链抗体片段，对其基因序列进行测序分析得到序列SEQ ID NO：1。
实施例2
SEQ ID NO：1所示基因序列编码的单链抗体片段的属性表征，具体步骤如下：
1.测试的核酸样品制备：核酸样品购自英骏生物技术有限公司，将核酸适量溶于磷酸钾的缓冲液中(ELISA缓冲液：pH7.4，50mM磷酸二氢钾，100mM KCl)，超微量紫外测定浓度，再用ELISA缓冲液稀释成5μM的DNA溶液在95℃下加热5min后缓慢冷却退火到室温，用圆二色谱法测定核酸的DNA二级结构及G-四链体构象。
测试的核酸样品代表性序列如表1所示，包括：
表1
Figure PCTCN2016101945-appb-000001
Figure PCTCN2016101945-appb-000002
GGGGAGGGTGGGGAGGGTGGGGAAGG/CCTTCCCCACCCTCCCCACCCTCCCCA为duplex序列，该duplex序列为双链序列，GGGGAGGGTGGGGAGGGTGGGGAAGG命名为duplex-1，序列号为SEQ ID NO：13，CCTTCCCCACCCTCCCCACCCTCCCCA命名为duplex-2，序列号为SEQ ID NO：14。
2.单链抗体片段的表达
通过基因工程技术将SEQ ID NO：1所示的基因序列插入到原核表达载体pSANG10中，获得重组质粒，经测序验证序列正确。再将重组质粒转化到大肠杆菌(BL21DE3)中进行表达与纯化：将储存在-80℃的菌种接种到5mL 2×TY+2％葡萄糖+50ng/μl卡那霉素的培养基中，放入摇床中30℃，200rpm过夜培养；取3mL过夜培养的菌种接种到300mL 2×TY+0.1％葡萄糖+50ng/μl卡那霉素的培养基中，放入摇床中37℃，250rpm培养3小时。加入IPTG到培养基中使其终浓度为0.2mM，放入摇床中25℃，280rpm过夜培养。收集过夜培养的细菌，4℃ 4000rpm离心30分钟，弃去上清，得菌体沉淀。用12mL的TES重悬菌体沉淀，冰上放置10分钟。加入18mL 5倍稀释的TES(50mM Tris-HCl,1mM EDTA,20％sucrose,pH8.0)，冰上放置15分钟。4℃ 8000rpm离心10分钟，取上清液。上清液用镍柱纯化，上样之后，用20mL洗涤液(PBS+100mM NaCl+10mM咪唑,pH8.0)洗去杂蛋白。待杂蛋白冲洗完全，用洗脱液(PBS+250mM咪唑,pH8.0)将抗体洗脱下来。洗脱下来的样品用超滤管超滤脱盐浓缩。浓缩后的样品用BCA法测定抗体浓度并用液氮速冻储存于-80℃。
取表达纯化过程中各个阶段的样品进行SDS-PAGE分析，得到结果如下图1所示，洗脱下来的样品即为目的抗体片段，并且纯度高。纯化获得的单链抗体片段可用于后续的活性表征。
3.单链抗体片段识别正平行型G-四链体DNA检测：
(1)体外酶联免疫吸附法检测
体外酶联免疫吸附试验(ELISA)方法如下：配制生物素标记的100nM待测G-四链体样品，分别结合到链霉亲和素包被的96孔板，4℃过夜。ELISA缓冲液洗板3次。用封闭液(ELISA缓冲液中加入3％的BSA)室温孵育3小时。ELISA缓冲液洗板3次。加入100μL封闭液稀释的正平行型G-四链体抗体片段，孵育1小 时后，用含0.1％Tween20的ELISA缓冲液洗板3次。加入用封闭液以1:5000稀释的HRP-Protein A，孵育1小时后，再用含0.1％ Tween20的ELISA缓冲液洗板3次。加入100μL新鲜配制的TMB底物，显色5～30分钟。加入50μL1mol/L H2SO4终止反应，并用酶标仪测定450nm各孔的吸光值。450nm处的吸光值代表抗体与DNA的结合强度，吸光值越高代表结合越强。
酶联免疫吸附法试验(ELISA)的结果显示(图2)，单链抗体高选择性结合正平行型G-四链体DNA(MYC和BCL-2)，对反平行型G-四链体(TBA)、混合型G-四链体(hTELO)、双链DNA、单链DNA以及发卡DNA结构未检测到结合，说明本发明所提的基因序列所编辑的单链抗体片段高选择性识别正平行型构象G-四链体DNA。
竞争性ELISA进一步验证抗体结合正平行型G-四链体的特异性，即在上述的ELISA实验方法加入抗体的步骤之前，将抗体与等量的竞争性DNA先孵育1个小时。检测结果显示(图3)，正平行型构象的G-四链体(MYC，KIT1，KIT2，HIF-1α，VEGF，KRAS，RET)可完全竞争结合单链抗体，而混合型G-四链体(hTELO)、反平行型G-四链体(TBA，HRAS-1)以及非G4序列(duplex，Random ssDNA，DNA Hairpin)都表现为很弱的竞争能力或无竞争能力。这一结果与之前的测定结果保持一致。
(2)细胞内免疫荧光法检测
接种数量为4×105的SiHa细胞于confocal专用皿中，过夜培养待细胞贴壁，用Lipo2000分别转染200nM的Cy5荧光基团标记的正平行型G-四链体DNA MYC以及反平行型G-四链体DNA TBA入SiHa细胞，培养24小时，收集细胞。用4％多聚甲醛室温避光固定15分钟，PBS洗5分钟，共洗3次，用0.3％Triton X-100(破膜)孵育室温孵育30分钟，PBS洗5分钟，3次，用5％山羊血清封闭液封闭细胞，室温孵育3小时，直接孵育大连抗体片段，4℃过夜，封闭液洗细胞样品，因单链抗体片段上携带FLAG标签，因此孵育抗FLAG的一抗标记scFv，4℃过夜，封闭液洗去未结合的抗体，同时孵育荧光标记的二抗以及DAPI(染细胞核)，室温孵育1小时，用封闭液洗去未结合的抗体及核染料后，使用激光共聚焦显微镜进行成像分析。
免疫荧光法的结果显示(图4)，本发明所提的基因序列编辑的单链抗体片段在细胞内显示的荧光点与荧光标记的正平行型G-四链体MYC的重叠融合高达80％，而与反平行型G-四链体TBA的荧光点重叠小于20％，表明，D1在细胞内对正平行型G-四链体的结合依然保持构象选择性，可用于细胞内正平行型构象G-四链体DNA的检测。
4.单链抗体片段与正平行型G-四链体抗体的结合常数测定
为定量表征本发明所提的基因序列编辑的单链抗体片段对不同构象G-四链体的亲和力，用ELISA的方法测定了scFv对MYC、TBA和hTELO的结合常数，结果显示(图5)，D1对正平行型构象G-四链体MYC的结合常数为27.8±0.9nM，而对反平行型G-四链体TBA以及混合型G-四链体hTELO检测不到结合。这一结果也同时验证本发明所提的基因序列编辑的单链抗体片段特异性结合正平行型构象G-四链体，并且结合亲和力高。
实施例3
将发明单链抗体应用于基因组正平行型G-四链体DNA的检测
为检测基因组正平行型G-四链体在活细胞内的形成并稳定存在，将SEQ ID NO：1所示的基因片段插入真核表达载体pEGFP-N3质粒中，通过瞬时转染的方法使单链抗体片段在活细胞中表达24小时。将表达了单链抗体片段的SiHa细胞收集，用1％多聚甲醛交联固定15分钟，终止交联裂解细胞，用Micrococcal Nuclease(Chip Grade)切断染色质得到200到500bp的染色质片段，该染色质片段去10％作为Input，剩下的进行后续的染色质免疫沉淀实验。将已经结合染色质上正平行型G-四链体DNA与scFv复合物用Protein A进行分离，最终得到scFv结合的来自人基因组的DNA片段。
将上述实验获得的DNA片段进行Hiseq2500测序分析，结果显示(图6)，相比于Input，本发明的单链抗体片段在活细胞内富集了8345个显著峰，显示正平行型G-四链体广泛分布在基因组中。单链抗体片段的结合位点主要分布于基因区(69.7％)，剩下的都分布在基因间区(30.3％)。正平行型G-四链体在基因区的分布情况为非翻译区UTR(5％),启动子转录起始位点promoter-TSS(16.8％),转录终止位点TTS(1.7％),外显子(14.7％)以及内含子(30.3％)。
为了验证本发明所提的基因序列所编辑的单链抗体片段在活细胞内结合的DNA片段是否为正平行型G-四链体DNA，从由scFv富集的前99个峰中选取寡核苷酸分别用圆二色谱法(CD)验证。结果显示(图7)，大部分的DNA片段形成了正平行型构象的G-四链体(91/99,92％)，剩下的DNA形成了混合型G-四链体，而在混合型G-四链体的CD图谱中也是以正平行型构象吸收峰为主峰。结果证明了本发明所提基因序列所编辑的单链抗体片段在基因组中选择性结合正平行型G-四链体的特性。
实施例4
将本发明单链抗体应用于端粒正平行型G-四链体DNA的检测
人端粒DNA由TTAGGG重复序列组成，符合形成G-四链体的序列特征，为了探究正平行型G-四链体结构是否在细胞内端粒区稳定形成，根据上述ChIP-seq数据，我们设定出现大于3个TTAGGG/CCCTAA重复序列的峰为端粒峰，进行进一步的生物信息学数据分析。结果显示(图8)，在scFv ChIP样品中，含有我们设定的端粒峰是Input样品中的4.8倍，说明细胞内端粒正平行型G-四链体被scFv检测到并被富集。同时，得到的样品做端粒dot blotting分析得到结果显示(图9)，scFv能够明显富集端粒序列，说明D1在活细胞内结合到正平行型端粒G-四链体，也同时说明这种二级结构的在活细胞内的形成并稳定存在。
已有文献报道，端粒富G序列在不同溶液背景下形成不同构象的G-四链体结构，我们选用了三种离子溶液环境下形成的三种不同构象端粒G-四链体以检测本发明的单链抗体片段对其结合能力。首先，我们通过CD实验验证了端粒富G序列在Na+离子溶液中退火形成反平行型G-四链体，在K+离子溶液中退火形成混合型G-四链体，在Sr2+溶液中，则形成正平行型G-四链体(图10)。在此三种溶液条件下，我们用ELISA实验分别检测scFv与三种不同构象端粒G-四链体的结合能力，结果显示(图11)，scFv与由Sr2+诱导的正平行型端粒G-四链体的结合常数为17.6±0.8nM，而与混合型以及反平行型的端粒G-四链体检测不到结合，此结果再一次说明了scFv与正平行型构象G-四链体结合的选择性，为细胞内端粒正平行型构象G-四链体的检测提供了可靠依据。
免疫荧光结果显示(图12)，相比于未处理细胞中的scFv与TRF2融合荧光点， 经RNase处理后的细胞中融合点并未发生显著变化，说明端粒区正平行型构象G-四链体DNA的存在。相应的经DNase处理后的细胞中融合点减少，也说明端粒区正平行型G-四链体DNA的形成并被本发明所提基因序列编辑的单链抗体片段特异性识别而检测到。
此外，我们将本发明设计的单链抗体(scFv)用于探究细胞内端粒G-四链体的主要存在构象，利用BG4与scFv这两个G4抗体进行了定量分析研究。抗体BG4能够结合不同构象的G-四链体DNA，而本发明所提基因序列编辑的scFv选择性结合正平行型构象的G4DNA，通过对比检测D1、BG4与端粒结合蛋白TRF2的共定位量来判断端粒G-四链体构象的唯一性或多样性。结果显示(图13)，在两组细胞端粒结合蛋白TRF2荧光点计数无显著变化的情况下，细胞核内BG4检测到的G-四链体的量明显多于scFv检测到的正平行型G-四链体的量，说明体内存在不同构象的G-四链体DNA，并且BG4检测到的端粒G-四链体的量也多于scFv检测到的正平行型G-四链体的量，但增加倍数不大(1.3倍)，说明端粒区除了正平行型构象G-四链体之外，还存在其他两种构象的G-四链体(混合型和反平行型)，而正平行型构象可能是端粒G-四链体在细胞内的主要存在方式(77％)。
上述结果表明本发明提供的单链抗体是一个优秀的大分子探针，可用于细胞内基因组以及特定功能区域的正平行型G-四链体DNA的结构研究。

Claims (7)

  1. 一种单链抗体，其特征在于，编码该单链抗体的基因序列如SEQ ID NO：1所示。
  2. 一种单链抗体的制备方法，其特征在于，将权利要求1所述的基因序列插入到原核表达载体中，获得重组质粒，再将重组质粒转化到大肠杆菌中进行表达与纯化。
  3. 权利要求1所述的单链抗体在特异性检测正平行型G-四链体中的应用。
  4. 权利要求1所述的单链抗体在制备特异性检测正平行型G-四链体材料中的应用。
  5. 一种特异性检测正平行型G-四链体的免疫方法，其特征在于，使用SEQ ID NO：1所示基因序列编码的单链抗体作为探针，用于特异性识别正平行型G-四链体。
  6. 根据权利要求5所述的免疫方法，其特征在于，所述的免疫方法为ELISA方法。
  7. 根据权利要求6所述的免疫方法，其特征在于，所述的免疫方法为ELISA方法时，具体步骤为：将生物素标记的待测核酸样品结合到链霉亲和素包被的96孔板，4℃过夜，采用ELISA缓冲液洗板，然后采用封闭液室温孵育，孵育后用ELISA缓冲液洗板；加入单链抗体，孵育后用ELISA缓冲液洗板，加入用封闭液稀释的HRP-Protein A，孵育后，用缓冲液洗板；然后加入TMB底物，显色，加入终止液终止反应，用酶标仪测定450nm的吸光值。
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