WO2023143043A1 - 一种融合表达Tau-4R蛋白的大肠杆菌工程菌株 - Google Patents

一种融合表达Tau-4R蛋白的大肠杆菌工程菌株 Download PDF

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WO2023143043A1
WO2023143043A1 PCT/CN2023/071534 CN2023071534W WO2023143043A1 WO 2023143043 A1 WO2023143043 A1 WO 2023143043A1 CN 2023071534 W CN2023071534 W CN 2023071534W WO 2023143043 A1 WO2023143043 A1 WO 2023143043A1
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protein
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蔡炯
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元本(珠海横琴)生物科技有限公司
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Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to an Escherichia coli engineering strain fusion expressing Tau-4R protein.
  • AD Alzheimer's disease
  • Alzheimer's disease is the most typical disease in dementia, accounting for about 55% of all dementia cases.
  • Alzheimer's disease shows two main pathological features of amyloid plaques and neurofibrillary tangles, the extracellular amyloid plaques are mainly composed of ⁇ -amyloid peptide, and the intracellular neurofibrillary tangles are mainly composed of Tau protein .
  • synaptic dystrophy, astrogliosis, microglial activation, and changes in mature neuronal markers were also included.
  • the clinical manifestations are cognitive impairment accompanied by memory decline.
  • the drugs currently used in the treatment of AD mainly include NMDA receptor antagonists (memantine) and acetylcholinesterase inhibitors (donepezil, galantamine), but these two types of drugs can only delay the symptoms of the disease, not Reverse the course of the disease.
  • a ⁇ -amyloid cascade hypothesis in Alzheimer's disease proposes that the accumulation of A ⁇ drives the onset of AD.
  • a ⁇ -associated mutations and the neurotoxicity of A ⁇ oligomers strongly support this hypothesis, A ⁇ plaques are also sometimes present in cognitively normal individuals brain, calling into question the A ⁇ cascade hypothesis.
  • tau aggregation is a major driver of neurodegenerative disease has only recently been widely accepted, hyperphosphorylated tau aggregates proliferate and spread from cell to cell, and tau pathology is more associated with clinical symptoms of dementia than A ⁇ pathology. have a better correlation.
  • ⁇ -amyloid antibody aducanumab (Biogen) is used to treat Alzheimer's disease and reduce the patient's amyloid- ⁇ plaque load, it also causes temporary swelling of the brain area and headache, confusion, and dizziness , vision changes, nausea, angioedema, urticaria, amyloid-related imaging abnormalities, headache, falls, diarrhea, and confusion.
  • Tau protein is a microtubule-associated protein involved in the development of the nervous system. It is enriched around the axons of neurons and combines with tubulin to form microtubules, which can stabilize microtubules and promote axonal transport. Under normal circumstances, the Tau gene encodes 16 exons, which are translated into Tau with a length of 352-441 amino acids and a molecular weight of 45-65 kDa. Tau protein can be divided into N-terminal projection functional region (N-terminal), proline-rich region, microtubule binding region (MBD) and C-terminal functional region. Among them, the length of the N-terminal is uncertain, and additional fragments from exons 2 (E2) and 3 (E3) can be inserted.
  • the proline-rich region contains a large number of phosphorylation sites, while the C-terminus provides some phosphorylation sites.
  • the repeat sequence (R1-R4) is located in MBD, which has the strongest microtubule binding force and promotes microtubule self-aggregation, and is also the core structure of double helical filaments (PHF).
  • Human Tau expresses 6 isoforms due to different mRNA editing methods, namely Tau352, 381, 383, 410, 412 and 441. The difference lies in whether there are 3 or 4 repeat sequences at the C-terminus and whether there are any 1 or 2 inserts.
  • Exon 10 encodes the R2 fragment, and Tau that can retain E10 and have 4 repeats is collectively referred to as 4RTau; Tau that does not retain E10 and has only 3 repeats is collectively referred to as 3RTau.
  • the expression levels of 3RTau and 4RTau in the adult brain are equivalent, while adult mice only express 4RTau.
  • Tau Under pathological conditions, Tau undergoes conformational changes, abnormal mRNA splicing, etc., dissociates from microtubules, undergoes a series of post-translational modifications, and forms pathological aggregation itself, which in turn causes neurodegeneration and eventually forms Tau lesions.
  • the inventors designed a fusion protein Tau-4R, which can be recombined into Escherichia coli for fusion expression. It is confirmed by experiments that the tau protein prepared by the bacterial strain can be used in the treatment research of Alzheimer's disease.
  • the invention provides a Tau-4R protein, characterized in that its amino acid sequence is shown in SEQ ID NO.1.
  • the present invention also provides a fusion protein, which is characterized in that the fusion protein includes the Tau-4R protein of the present invention.
  • the fusion protein comprises maltose binding protein (MBP) and Tau-4R protein of the present invention.
  • MBP maltose binding protein
  • amino acid sequence of the fusion protein is shown in SEQ ID NO.3.
  • the present invention also provides a derivative protein of the fusion protein, which is characterized in that the derivative protein of the fusion protein is added or partially replaced by a linking sequence on the basis of the fusion protein.
  • the linking sequence is GGGGSGGGGSGGGGS.
  • the connecting sequence (linker) of the present invention is a flexible short peptide sequence, which can increase the flexibility and make the reporter protein fragments easy to approach and form complementarity.
  • the derivative protein of the fusion protein has an amino acid sequence as shown in SEQ ID NO.4.
  • the present invention also provides a polynucleotide encoding the Tau-4R protein, the fusion protein and/or the derivative protein of the fusion protein of the present invention.
  • polynucleotide sequence is as shown in SEQ ID NO.2.
  • the present invention also provides a recombinant expression vector, which is characterized in that it comprises Tau-4R protein and/or its fusion protein, or a derivative protein of the fusion protein.
  • the present invention also provides a host cell, which is characterized in that it contains Tau-4R protein and/or its fusion protein, or a derivative protein of the fusion protein.
  • the fusion protein is obtained by inducing the expression strain of Tau-4R protein with MBP tag.
  • the fusion protein is induced by IPTG, and the strain is obtained by connecting and transforming a vector with an MBP tag and a substance containing the Tau-4 gene of the present invention.
  • the material containing the Tau-4 gene of the present invention may be a PCR product or a plasmid.
  • the vector with the MBP tag is selected from one or more of pMal-p5x, pMAL-c2G, pMAL-c2x, pMAL-c4x, pET-28b-MBP-His.
  • the present invention provides a pharmaceutical composition, which comprises the Tau-4R protein of the present invention and/or its fusion protein, or the derivative protein of the fusion protein.
  • the present invention also provides a preparation method of the above-mentioned Tau-4R protein.
  • the present invention also provides a method for preparing the above-mentioned fusion protein or its derivative protein, comprising the following steps:
  • step (2) Connecting the vector with the MBP tag to the substance containing the Tau-4R protein obtained in step (1), transforming and inducing expression to obtain the fusion protein.
  • the substance containing the Tau-4 gene of the present invention may be a PCR product or a plasmid.
  • the step (1) is obtained by molecular biology methods or chemical synthesis methods.
  • the vector with the MBP tag is selected from one or more of pMal-p5x, pMAL-c2G, pMAL-c2x, pMAL-c4x, pET-28b-MBP-His.
  • the fusion protein is induced by IPTG.
  • the present invention also provides a method for purifying the fusion protein, comprising the following steps: using an amylose-resin affinity column to purify the fusion protein.
  • the ion exchange column is used for elution.
  • the protein purified by the amylose-resin affinity column is eluted with 20mMTris-HCl (pH7.4), go to the Q-Sepharose FF ion exchange column, and then wash with 20mMTris-HCl, 200mMNaCl (pH7.4) take off.
  • the present invention also provides the use of Tau-4R protein, fusion protein or derivative protein of said fusion protein in the preparation of neurological disease medicine.
  • the neurological disease is a neurodegenerative disease, more preferably Alzheimer's disease.
  • the present invention also provides the application of Tau-4R protein, fusion protein or derivative protein of said fusion protein in medicine for prolonging the life of patients with neurological diseases.
  • the neurological disease is a neurodegenerative disease, more preferably, Alzheimer's disease.
  • amino acid sequence of wherein said fusion protein is as shown in SEQ ID NO.3.
  • amino acid sequence of the derivative protein of the fusion protein is shown in SEQ ID NO.4.
  • the inventors designed a fusion protein Tau-4R, which can be recombined into Escherichia coli to achieve fusion expression. After being purified by an affinity column and an ion exchange column, the purity is greatly improved and the production cost is greatly saved.
  • tau protein prepared by the strain can be used in the treatment research of Alzheimer's disease.
  • FIG. 1 Tau-4R gene double enzyme digestion identification (1. double enzyme digestion product; M: Marker10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700 , 600, 500, 400, 300, 200, 100bp).
  • FIG. 1 Sequencing map of Tau-4R gene.
  • FIG. 4 Purification of cell fusion expression Tau-4R protein (1: Before induction; 2: After induction; 3: Precipitation; 4: Precipitation after ultrasound; 5: Cell supernatant before loading; 6: Cell supernatant Permeate solution; 7-8: wash off bacteria; 9: low molecular weight protein Marker, 97.4, 66.2, 43, 31, 22.0, 14.4KD).
  • Figure 7 Preservation of fusion expressed Tau-4R protein (1: Marker, 97.4, 66.2, 43, 31, 22.0, 14.4KD; 2: 4°C after lyophilization; 3: -20°C; 4: -80°C preservation; 5: before freeze-drying).
  • Fig. 9 Effect of TauVax immunization on three-transformed AD mice on the stage jumping latency (stage time).
  • FIG. 10 TauVax immunization of triple-transformed AD mice can reduce the amyloid plaque load in the hippocampus (A. wild-type mice; B. triple-transformed mice treated with saline; C. triple-transformed mice immunotherapy with TauVax protein ).
  • amino acid sequence SEQ ID NO.1 of the designed recombinant protein Tau-4R is as follows: TENLKHQPGGGKGSKDNIKHVPGGGSGSLGNIHHKPGGGQGSLDNITHVPGGGN.
  • the following gene was designed by optimizing codon preference in Escherichia coli.
  • the sequence SEQ ID NO.2 is as follows: CATATGACTGAAAATCTGAAACATCAACCTGGCGGCGGTAAAGGCTCTAAAGATAACATCAAGCACGTTCCTGGTGGCGGTTCTGGTTCCCTGGGTAACATCCATCACAAACCGGGTGGCGGTCAAGGTAGCCTGGATAATATCACTCATGTGCCAGGTGGTGGCAATCC ATGG.
  • the 5' end of the gene has an NdeI restriction site, and the 3' end has an NcoI restriction site.
  • Example 2 Establishment of an Escherichia coli strain expressing Tau-4R protein in fusion
  • the pUCm-T-Tau-4R and pMal-p5x plasmids were digested with NdeI and NcoI, and the small and large fragments were collected, ligated with T4 ligase, transformed into DH5 ⁇ competent bacteria, and screened with ampicillin LB plate. Then PCR was used to identify and extract the plasmid for double digestion with NdeI and NcoI to determine the size of the fragment between 100-200bp to obtain the pMal-p5x-Tau-4R plasmid. Two clones were randomly selected and transferred to ampicillin LB medium for overnight culture at 37°C and 220rpm.
  • DH5 ⁇ -pMal-p5x-Tau-4-1 and DH5 ⁇ -pMal-p5x-Tau-4-2 strains had nascent protein expression around 50KD; and DH5 ⁇ -pMal-p5x had nascent protein expression at 43KD, establishing Escherichia coli strains DH5 ⁇ -pMal-p5x-Tau-4-1 and DH5 ⁇ -pMal-p5x-Tau-4-2 expressing Tau-4R protein were successfully fused.
  • Bacterial culture supernatant was filtered through a membrane (first passed 0.45 ⁇ m, then 0.22 ⁇ m), passed through an amylose-resin column, eluted with 10 mM maltose, 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA; pH 7.4, and the obtained protein was subjected to SDS-PAGE As a result, a protein of about 50 KD was purified (Fig. 5). The purity of the target protein obtained in the culture medium was 60%.
  • amino acid sequence SEQ ID NO.3 of MBP-Tau-4R is as follows: KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSAL MFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMP NIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSNNNNNNNNNNLGIEGRISHMTENLKHQPGGGKGSKDNIKHVPGGGSGSLGNI
  • sequence SEQ ID NO.4 is as follows:
  • the MBP-Tau-4R purified by the amylose-resin affinity column was dialyzed with 20mMTris-HCl (pH7.4), put on the Q-Sepharose FF ion exchange column, and then eluted with 20mMTris-HCl and 200mMNaCl (pH7.4) to obtain The protein purity is higher ( Figure 6).
  • the MBP-Tau-4R protein was lyophilized and stored at 4°C for 1 month, and the unlyophilized samples stored at -20 and -80°C for 1 month were analyzed by SDS-PAGE to determine the purity of the target protein.
  • the MBP-Tau-4R protein concentration was adjusted to 1 mg/ml, mixed with an equal amount of 2% aluminum hydroxide adjuvant to obtain TauVax.
  • 15-month-old triple-transformed mice (APP/PS1/Tau) were immunized with TauVax, and 100 ⁇ L was injected intramuscularly, twice a week for 3 consecutive weeks.
  • the experiment was divided into three groups. The first group consisted of 6 15-month-old normal mice C57BL/6J injected with normal saline; the second group consisted of 6 15-month-old triple-transformed mice injected with normal saline; the third group consisted of 6 15 Three-month-old mice (APP/PS1/Tau) were injected with TauVax.
  • Example 8 The platform test of MBP-Tau-4R protein after immunization of triple-transferred mice
  • the size of the four reaction boxes on the mouse jumping platform is 25cm ⁇ 25cm ⁇ 30cm, and the bottom of the box is a copper grid for continuous electrical stimulation of 36V.
  • a rubber mat with a diameter of 12 cm and a height of 4.5 cm was placed in the middle of each room as a safe area for mice to avoid electric shocks.
  • the mice were adapted to the environment for 3 minutes before the experiment, and then the copper grid at the bottom was supplied with 36V alternating current. Record the reaction time of the mouse jumping on the rubber mat after receiving the electrical stimulation, as the academic performance.
  • the mouse was placed in the platform diving apparatus for 3 minutes to adapt again, and placed on the rubber mat, and the latency period for jumping on the rubber mat after stepping off the platform for the first time was recorded as a memory score.
  • the results showed that in the first test and the second memory recall test, the stage latency of the mice in the model group was significantly longer than that of the mice in the normal group.
  • TauVax treatment the onset latency of the mice in the treatment group was shortened ( Figure 9), suggesting that the learning and memory abilities of the three-turn mice decreased, while TauVax treatment improved the learning and memory abilities of the three-turn mice.
  • mice were perfused after the behavioral testing.
  • the heart was perfused, and the brain tissue was taken, fixed in 4% paraformaldehyde in a refrigerator at 4°C for 24 hours; on the second day, the tissue was taken out from 4% paraformaldehyde, washed with tap water overnight;
  • Mark the embedding box with a pencil pass through an alcohol tank (concentration is 50%-75%-85%-95%-100%-TO transparent agent), 1 hour for each tank, and pass through a paraffin tank (I wax-II Wax-III wax, wax I is the oldest, wax III is the newest, put the wax in an oven at 65°C for 6-8h in advance, 30min each, and finally embed in paraffin, store in the refrigerator at 4°C, slice (4um) on the fourth day- Baked slices (65°C, 2h), TO transparent agent transparent (100%-95%-85%-75%-50%, alcohol dissolved, 10min for each jar), 97.5°C antigen restoration for 40min (EDTA citrate restoration solution

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Abstract

本发明公开了一种融合蛋白Tau-4R，该蛋白可以重组到大肠杆菌中实现融合表达。通过实验证实，该菌株制备的tau蛋白有可能用于阿尔茨海默的治疗研究。

Description

一种融合表达Tau-4R蛋白的大肠杆菌工程菌株
本申请要求享有2022年1月29日向中国国家知识产权局提交的，专利申请号为2022101129048，发明名称为“一种融合表达Tau-4R蛋白的大肠杆菌工程菌株”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及生物医药领域，具体而言，本发明涉及一种融合表达Tau-4R蛋白的大肠杆菌工程菌株。
背景技术
阿尔茨海默病(AD)是神经退行性疾病，在老年人群中发病率极高，发病人口逐渐增长。2020年，全球大约有6000万名AD患者，预计到2050年，AD患者的人数将达到1.5亿左右。
阿尔茨海默病是痴呆中最典型的疾病,占所有痴呆病例的55％左右。阿尔茨海默病表现出淀粉样斑块和神经纤维缠结两个主要的病理特征，细胞外的淀粉样斑块主要由β淀粉样肽组成，细胞内的神经纤维缠结主要由Tau蛋白组成。此外还包括神经突触营养不良、星形胶质细胞增生、小胶质细胞活化以成熟神经元标记物改变等。临床表现为认知能力出现障碍并伴随记忆能力衰退。目前应用于治疗AD的药物主要包括NMDA受体拮抗剂(美金刚)和乙酰胆碱酯酶抑制剂(多奈哌齐、加兰他敏)两类药物，但是这两类药物只能延缓疾病的症状，并不能逆转疾病的进程。
阿尔茨海默病的β淀粉样级联假说认为Aβ的积累驱动AD发病.尽管Aβ相关突变和Aβ寡聚物的神经毒性强烈支持该假说，但Aβ斑块有时也出现在认知正常的人脑中，引起了人们对Aβ级联假说的质疑。而tau蛋白聚集是神经退行性疾病的主要驱动力这一假说最近才被广泛接受，过磷酸化tau蛋白聚集体在细胞之间增殖和传播，tau病理学与痴呆症的临床症状比Aβ病理学具有更好的相关性。虽然美国FDA加速批准的β淀粉样蛋白抗体aducanumab(Biogen)用于治疗阿兹海默症，降低患者的淀粉样β斑块负载，但是也会出现大脑区域的暂时性肿胀和头痛、困惑、眩晕、视力改变、恶心、血管性水肿、荨麻疹、淀粉样蛋白相关成像异常、头痛、跌倒、腹泻和精神错乱等副作用。
Tau蛋白是一种参与神经系统发育的微管相关蛋白，富集于神经元轴突周围，与微管蛋白结合形成微管，可稳定微管并促进轴突运输。正常情况下，Tau基因编码16个外显子，翻译为 长352～441个氨基酸，分子量为45～65kDa的Tau。Tau蛋白可分为N末端投射功能区(N端)、脯氨酸富集区、微管结合区(MBD)和C末端功能区。其中，N端长度不确定，可插入由外显子2(E2)、3(E3)额外片段。脯氨酸富集区含大量磷酸化位点，而c端提供了部分磷酸化位点。重复序列(R1-R4)位于MBD，其微管结合力最强并促进微管自聚集，同时也是双螺旋细丝(PHF)的核心结构。人类Tau因mRNA剪辑方式不同，表达出6种同工异构体，即Tau352、381、383、410、412和441，其差异在于c端是否存在3或4个重复序列，N端是否存在有1或2个插入物。外显子10(E10)编码R2片段，能保留E10且具有4个重复序列的Tau统称为4RTau；不保留E10且只有3个重复序列的Tau统称为3RTau。成年人脑内3RTau、4RTau表达量相当，而成年鼠只表达4RTau。
病理条件下，Tau经历构象改变、异常mRNA剪接等，从微管解离，经系列翻译后修饰，自身形成病理性聚集，进而造成神经退行性病变，最终形成Tau病变。
发明内容
为了防止或逆转tau蛋白的寡聚化，本发明人设计出一种融合蛋白Tau-4R，该蛋白可以重组到大肠杆菌中实现融合表达。通过实验证实，该菌株制备的tau蛋白能用于阿尔茨海默的治疗研究。
为此，本发明采用的技术方案如下：
本发明提供了一种Tau-4R蛋白，其特征在于，其氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种融合蛋白，其特征在于，所述融合蛋白包括本发明的Tau-4R蛋白。优选地，所述融合蛋白包含麦芽糖结合蛋白(MBP)和本发明的Tau-4R蛋白。
根据本发明，所述融合蛋白的氨基酸序列如SEQ ID NO.3所示。
本发明还提供了所述融合蛋白的衍生蛋白，其特征在于，所述融合蛋白的衍生蛋白是在融合蛋白的基础上增加或部分替换为连接序列。优选地，所述连接序列为GGGGSGGGGSGGGGS。
本发明的连接序列(linker)，是一种柔性短肽序列，起到增加柔性的作用,使报告蛋白片断之间易于接近形成互补。
根据本发明，所述融合蛋白的衍生蛋白，其氨基酸序列如SEQ ID NO.4所示。
本发明还提供了一种多核苷酸，其编码本发明的Tau-4R蛋白、融合蛋白和/或所述融合蛋白的衍生蛋白。
优选地，所述多核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种重组表达载体，其特征在于，其包含Tau-4R蛋白和/或其融合蛋白，或所述融合蛋白的衍生蛋白。
本发明还提供一种宿主细胞，其特征在于，其包含Tau-4R蛋白和/或其融合蛋白，或所述融合蛋白的衍生蛋白。
根据本发明，所述融合蛋白通过诱导带有MBP标签的Tau-4R蛋白表达菌株获得。
优选地，所述融合蛋白是通过IPTG诱导的，所述菌株是通过带有MBP标签的载体与含本发明的Tau-4基因的物质连接，转化得到。其中，所述含本发明的Tau-4基因的物质可以是PCR产物或质粒。所述带有MBP标签的载体选自pMal-p5x、pMAL-c2G、pMAL-c2x、pMAL-c4x、pET-28b-MBP-His中的一种或多种。
本发明提供一种药物组合物，其包含本发明的Tau-4R蛋白和/或其融合蛋白，或所述融合蛋白的衍生蛋白。
本发明还提供上述Tau-4R蛋白的制备方法。
本发明还提供上述融合蛋白或其衍生蛋白的制备方法，包括如下步骤：
(1)获得含有本发明的Tau-4R蛋白基因的物质；
(2)将带有MBP标签的载体和步骤(1)获得的含有Tau-4R蛋白的物质连接，转化诱导表达，获得所述融合蛋白。
根据本发明，优选地，所述含有本发明的Tau-4基因的物质可以是PCR产物或质粒。其中，优选地，所述步骤(1)，通过分子生物学方法或化学合成的方法获得。所述步骤(2)中，所述带有MBP标签的载体选自pMal-p5x、pMAL-c2G、pMAL-c2x、pMAL-c4x、pET-28b-MBP-His中的一种或多种。所述融合蛋白是通过IPTG诱导的。
本发明还提供了融合蛋白的纯化方法，包括如下步骤：使用amylose-resin亲和柱纯化的所述融合蛋白。优选地，洗脱之后，再上离子交换柱洗脱。
根据本发明，将amylose-resin亲和柱纯化的蛋白，用20mMTris-HCl(pH7.4)洗脱后，上Q-Sepharose FF离子交换柱，再用20mMTris-HCl、200mMNaCl(pH7.4)洗脱。
本发明还提供了Tau-4R蛋白、融合蛋白或所述融合蛋白的衍生蛋白在制备神经性疾病药物中的用途。
根据本发明，所述神经性疾病为神经退行性疾病，更优选地，为阿尔茨海默病。
本发明还提供了Tau-4R蛋白、融合蛋白或所述融合蛋白的衍生蛋白在延长神经性疾病患者寿命的药物中的用途。优选地，所述神经性疾病为神经退行性疾病，更优选地，为阿尔茨海默病。
根据本发明的用途，其中所述Tau-4R蛋白的氨基酸序列如SEQ ID NO.1所示。
根据本发明的用途，其中所述融合蛋白的氨基酸序列如SEQ ID NO.3所示。
根据本发明的用途，其中所述融合蛋白的衍生蛋白的氨基酸序列如SEQ ID NO.4所示。
本发明的有益效果：
1、本发明人设计出一种融合蛋白Tau-4R，该蛋白可以重组到大肠杆菌中实现融合表达。经过亲和柱和离子交换柱纯化后，大幅度提高了纯度，大大节约了生产成本。
2、通过实验证实，该菌株制备的tau蛋白能用于阿尔茨海默的治疗研究。
附图说明：
图1.Tau-4R基因双酶切鉴定(1.双酶切产物；M:Marker10000、8000、6000、5000、4000、3500、3000、2500、2000、1500、1200、1000、900、800、700、600、500、400、300、200、100bp)。
图2.Tau-4R基因测序图。
图3.融合表达Tau-4R蛋白的大肠杆菌菌株的鉴定(M：低分子量蛋白质标准97.4、66.2、43、31、22、14.4kD；
1：IPTG诱导前的DH5α-pMal-p5x-Tau-4-1菌株；
2：IPTG诱导后的DH5α-pMal-p5x-Tau-4-1菌株；
3：IPTG诱导前的DH5α-pMal-p5x-Tau-4-2菌株；
4：IPTG诱导后的DH5α-pMal-p5x-Tau-4-2菌株；
5：IPTG诱导前的DH5α-pMal-p5x菌株；
6：IPTG诱导后的DH5α-pMal-p5x菌株。
图4.菌体融合表达Tau-4R蛋白的纯化(1：诱导前；2：诱导后；3：沉淀；4：超声后沉淀；5：菌体上清上样前；6：菌体上清穿出液；7-8：菌体上清洗脱；9：低分子量蛋白Marker，97.4，66.2，43，31，22.0，14.4KD)。
图5.培养基融合表达Tau-4R蛋白的纯化(1：培养基上样前；2：上样后；3：Marker，97.4，66.2，43，31，22.0，14.4KD；4：洗脱液)。
图6.融合表达Tau-4R蛋白的流程优化(1：Marker，97.4，66.2，43，31，22.0，14.4KD；2：洗脱液)。
图7.融合表达Tau-4R蛋白的保存(1：Marker，97.4，66.2，43，31，22.0，14.4KD；2：冻干后4℃保存；3：-20℃保存；4：-80℃保存；5：冻干前)。
图8.TauVax免疫三转AD小鼠可以缩短第四次水迷宫探索的潜伏期(上台时间)。
图9.TauVax免疫三转AD小鼠对跳台上台潜伏期(上台时间)的影响。
图10.TauVax免疫三转AD小鼠可以降低海马的淀粉样斑块负载(A.野生型小鼠；B.用生理盐水治疗的三转小鼠；C.TauVax蛋白免疫治疗的三转小鼠)。
图11.Tau-4R免疫降低三转小鼠大脑海马Tau蛋白磷酸化。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1.设计基因
设计重组蛋白质Tau-4R的氨基酸序列SEQ ID NO.1如下：TENLKHQPGGGKGSKDNIKHVPGGGSGSLGNIHHKPGGGQGSLDNITHVPGGGN。
通过大肠杆菌偏好密码子优化，设计以下基因,序列SEQ ID NO.2如下：CATATGACTGAAAATCTGAAACATCAACCTGGCGGCGGTAAAGGCTCTAAAGATAACATCAAGCACGTTCCTGGTGGCGGTTCTGGTTCCCTGGGTAACATCCATCACAAACCGGGTGGCGGTCAAGGTAGCCTGGATAATATCACTCATGTGCCAGGTGGTGGCAATCCATGG。基因的5’端带有NdeI酶切位点，3’端带有NcoI酶切位点。通过多重PCR的方法合成，转化入pUCm-T质粒，通过氨苄青霉素LB平板进行筛选，经过IPTG/X-gal染色挑选白色菌落进行PCR鉴定，再通过PCR鉴定、提取质粒进行NdeI、NcoI双酶切，确定片段大小在100-200bp之间，和理论值168bp接近(图1)
送酶切鉴定正确的质粒进行测序，结果显示碱基序列完全正确，长度大小正确，在5’端具有NdeI酶切位点，3’端带有NcoI酶切位点(图2)
实施例2.融合表达Tau-4R蛋白的大肠杆菌菌株建立
将pUCm-T-Tau-4R、pMal-p5x质粒用NdeI、NcoI双酶切，分别收集小片段和大片段质粒，用T4连接酶连接，转化DH5α感受态细菌，用氨苄青霉素LB平板进行筛选，再用PCR鉴定、提取质粒进行NdeI、NcoI双酶切，确定片段大小在100-200bp之间，获得 pMal-p5x-Tau-4R质粒。随机挑选两个克隆转入到氨苄青霉素LB培养基在37℃、220rpm过夜培养。第二天1：1000转入5ml的氨苄青霉素LB培养基中，37℃、220rpm培养2.5小时后加入1mM的IPTG过夜诱导，以pMal-p5x空质粒转化感受态DH5α的菌株为阴性对照。随后进行SDS-PAGE分析。
结果发现的DH5α-pMal-p5x-Tau-4-1、DH5α-pMal-p5x-Tau-4-2菌株在50KD附近有新生蛋白质表达；而DH5α-pMal-p5x在43KD处有新生蛋白质表达，建立成功融合表达Tau-4R蛋白的大肠杆菌菌株DH5α-pMal-p5x-Tau-4-1、DH5α-pMal-p5x-Tau-4-2。
实施例3.MBP-Tau-4R蛋白质纯化
通过1mM IPTG诱导DH5α-pMal-p5x-Tau-4-1菌株得到含MBP-Tau-4R蛋白质的菌泥，用20mMTris-HCl、200mMNaCl、1mM EDTA；pH7.4重悬后超声破碎，12000rpm、10min离心3次后过0.45μm、0.22μm滤膜，上amylose-resin柱，用10mM麦芽糖、20mMTris-HCl、200mMNaCl、1mM EDTA；pH7.4洗脱，得到的蛋白质进行SDS-PAGE分析。
结果纯化出约50KD的蛋白质，和MBP-Tau-4R蛋白质理论分子量一致，但是，目标蛋白质纯度只有34％(图4)。
细菌培养上清过滤膜(先过0.45μm，再过0.22μm)，过amylose-resin柱，用10mM麦芽糖、20mMTris-HCl、200mMNaCl、1mM EDTA；pH7.4洗脱，得到的蛋白质进行SDS-PAGE分析，结果约50KD的蛋白质被纯化出来(图5)。培养基中得到的目的蛋白纯度较高，为60％。
MBP-Tau-4R的氨基酸序列SEQ ID NO.3如下：KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISHMTENLKHQPGGGKGSKDNIKHVPGGGSGSLGNIHHKPGGGQGSLDNITHVPGGGNPWAAAISSTDPNSLQVIK。
实施例4.MBP-Tau-4R蛋白质的衍生
将NNNNNNNNNN替换为GGGGSGGGGS得到新的衍生分子，序列SEQ ID NO.4如下：
Figure PCTCN2023071534-appb-000001
实施例5.MBP-Tau-4R蛋白质纯化流程的优化
将amylose-resin亲和柱纯化的MBP-Tau-4R用20mMTris-HCl(pH7.4)透析，上Q-Sepharose FF离子交换柱，再用20mMTris-HCl、200mMNaCl(pH7.4)洗脱，得到的蛋白质纯度更高(图6)。
实施例6.MBP-Tau-4R蛋白质保存条件筛选
将MBP-Tau-4R蛋白冻干，4℃保存1个月，与-20、-80℃保存1个月未经冻干的样本用SDS-PAGE分析，测定其目的蛋白质纯度。
表1.不同保存条件对融合表达Tau-4R蛋白纯度的影响
Figure PCTCN2023071534-appb-000002
如图7和表1所示，未见MBP-Tau-4R蛋白纯度明显下降。
实施例7.MBP-Tau-4R蛋白质的免疫接种三转小鼠和水迷宫行为学测试
将MBP-Tau-4R蛋白质浓度调节到1mg/ml，和等量2％的氢氧化铝佐剂混合，得到TauVax。用TauVax免疫15月龄三转小鼠(APP/PS1/Tau)，肌肉注射100μL，每周2次，连续3周。实验分三组，第一组为6只15月龄正常小鼠C57BL/6J，注射生理盐水；第二组为6只15月龄三转小鼠，注射生理盐水；第三组为6只15月龄三转小鼠(APP/PS1/Tau)，注射TauVax。实验结束后进行水迷宫行为学检测，结果显示：TauVax免疫三转AD小鼠第二次水迷宫探索的潜伏期影响不大，但可以缩短第四次水迷宫探索的潜伏期，从46.7±3.2降低34.8±7.9s(p<0.05)(图8)。 提示注射TauVax可以显著改善三转小鼠的学习能力。
实施例8.MBP-Tau-4R蛋白质的免疫三转小鼠后的跳台测试
小鼠跳台四间反应箱规格为25cm×25cm×30cm，箱底是通36V连续电刺激的铜栅。每间中间放置一直径12cm、高4.5cm的橡皮垫，作为小鼠回避电击的安全区。小鼠实验前先适应环境3min，然后底部铜栅通以36V交流电。记录小鼠受到电刺激后跳上橡皮垫的反应时间，作为学习成绩。24h后将小鼠再次置于跳台仪中适应3min，将其置于橡皮垫上，记录第1次下台后跳上橡皮垫的潜伏期，作为记忆成绩。结果表明，第1次测试和第2次记忆重现测试中，模型组小鼠的上台潜伏期明显比正常组小鼠延长。经TauVax治疗后，治疗组小鼠的上台潜伏期缩短(图9)，提示：三转鼠的学习和记忆能力下降，而TauVax治疗改善了三转鼠的学习和记忆能力。
实施例9.MBP-Tau-4R蛋白质的免疫三转小鼠后的免疫组织化学染色
行为检测完后灌流小鼠。第一天心脏灌流，取脑组织，4％多聚甲醛4℃冰箱固定24h；第二天组织从4％多聚甲醛中取出，冲自来水过夜；第三天将组织切成适合的大小，并用铅笔在包埋盒上做好标记，过酒精缸(浓度为50％-75％-85％-95％-100％-TO透明剂)，每个缸子1小时，过石蜡缸(I蜡-II蜡-III蜡，I蜡最旧，III蜡最新，提前把蜡放入65℃烘箱6-8h)，每个30min，最后包埋于石蜡,4℃冰箱保存，第四天切片(4um)-烤片(65℃，2h)，TO透明剂透明(100％-95％-85％-75％-50％，酒精溶解，每个缸子10min)，97.5℃抗原修复40min(EDTA柠檬酸盐修复液)，然后加内源性过氧化物酶阻断剂(10％过氧化氢)盖住玻片、室温孵育10min阻断，PBS洗涤3次，每次5min。除去PBS，把玻片放置在载玻片上，每张切片滴加正常山羊血清工作液(1％正常山羊血清)，室温孵育10-15min。去除血清，滴加一抗6E10(amyloid1-16)或者AT8(S 202T 205)，4℃过夜；第五天PBS洗涤3次，加入二抗(生物素标记山羊抗小鼠lgG)，37℃孵育60min。PBS洗涤3次，滴加辣根酶标记链亲和素(与二抗结合)室温孵育10min，PBS洗3次，5min/次。除去PBS，滴加新鲜DAB溶液(1-2min完成，切片变黄立即终止)。自来水浸泡1-2min，苏木素复染1min。1％盐酸酒精过一下2-3s(去掉非特异性染色)，自来水浸泡返蓝约15-30min。梯度酒精脱水(50％-75％-85％-95％-100％)-TO透明剂-封片剂封片；第六天片子干后显微镜观察拍照。结果显示三转小鼠在大脑海马区出现大量的淀粉样斑块，而经过TauVax治疗后，淀粉样斑块明显减少(图10)。同时显示和野生型小鼠相比，三转小鼠大脑海马出现大量磷酸化Tau，DAB染色面积从3.27±1.07％上升到18.47±0.92％(p<0.0001)；经过TauVax治疗后，磷酸化Tau显著减少，DAB染色面积从18.47±0.92％下降到10.63±1.36％(p<0.01)(图11)。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。

Claims (10)

  1. 一种Tau-4R蛋白，其特征在于，其氨基酸序列如SEQ ID NO.1所示。
  2. 一种融合蛋白或其衍生蛋白，其特征在于，所述融合蛋白包括权利要求1所述的Tau-4R蛋白。
    优选地，所述融合蛋白包含MBP和权利要求1所述的Tau-4R蛋白；
    优选地，所述融合蛋白的氨基酸序列如SEQ ID NO.3所示；
    所述融合蛋白的衍生蛋白是在融合蛋白的基础上增加或部分替换为连接序列；优选地，所述连接序列为GGGGSGGGGSGGGGS；
    优选地，所述融合蛋白的衍生蛋白，其氨基酸序列如SEQ ID NO.4所示。
  3. 一种多核苷酸，其特征在于，所述多核苷酸编码权利要求1所述的Tau-4R蛋白、权利要求2所述融合蛋白或其衍生蛋白。
    优选地，其中，所述编码权利要求1所述的Tau-4R蛋白的多核苷酸序列如SEQ ID NO.2所示。
  4. 表达权利要求1所述的Tau-4R蛋白、或权利要求2所述融合蛋白或其衍生蛋白的基因的重组表达载体或宿主细胞；
    优选地，所述Tau-4R蛋白的基因序列如SEQ ID NO.2所示。
  5. 一种药物组合物，特征在于包含权利要求1所述的Tau-4R蛋白、或权利要求2所述融合蛋白或其衍生蛋白；
    优选地，所述药物组合物含有药学上可接受的载体或佐剂；
    进一步优选地，所述佐剂优选氢氧化铝佐剂。
  6. 权利要求1所述的Tau-4R蛋白、权利要求2所述融合蛋白和/或权利要求3所述融合蛋白的衍生蛋白的制备方法。
  7. 权利要求6所述的的制备方法，其中所述融合蛋白的制备方法，包括如下步骤：
    (1)获得含有权利要求1所述的Tau-4R蛋白基因的物质；
    (2)将带有MBP标签基因的载体和步骤(1)获得的含有Tau-4R蛋白基因的物质连接，转化，诱导表达，获得所述融合蛋白；
    优选地，所述含有权利要求1所述的Tau-4R蛋白基因的物质可以是PCR产物或质粒；其中，优选地，所述步骤(1)，通过分子生物学方法或化学合成的方法获得所述的Tau-4R蛋白基因的物质；
    优选地，所述步骤(2)中，所述带有MBP标签的载体选自pMal-p5x、pMAL-c2G、pMAL-c2x、pMAL-c4x、pET-28b-MBP-His中的一种或多种；
    优选地，所述融合蛋白通过IPTG诱导表达。
  8. 权利要求2所述融合蛋白或其衍生蛋白的纯化方法，包括如下步骤：
    使用amylose-resin亲和柱纯化的所述融合蛋白；
    优选地，洗脱之后，上离子交换柱洗脱；更优选地，将amylose-resin亲和柱纯化的蛋白，用20mMTris-HCl(pH7.4)洗脱后，上Q-Sepharose FF离子交换柱，再用20mMTris-HCl、200mMNaCl(pH7.4)洗脱。
  9. 权利要求1所述的Tau-4R蛋白、权利要求2所述融合蛋白或其衍生蛋白在制备治疗神经性疾病药物中的用途；
    优选地，所述神经性疾病为神经退行性疾病，更优选地，为阿尔茨海默病。
  10. 权利要求1所述的Tau-4R蛋白、权利要求2所述融合蛋白或其衍生蛋白在制备延长神经性疾病患者寿命的药物中的用途；
    优选地，所述神经性疾病为神经退行性疾病，更优选地，为阿尔茨海默病。
PCT/CN2023/071534 2022-01-29 2023-01-10 一种融合表达Tau-4R蛋白的大肠杆菌工程菌株 WO2023143043A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104185640A (zh) * 2011-09-19 2014-12-03 阿克松神经系统科学公司 阿尔茨海默病中的tau蛋白介导病变的基于蛋白质的疗法和诊断
WO2019084488A1 (en) * 2017-10-27 2019-05-02 United Neuroscience IMMUNOGENIC CONSTRUCTS OF PEPTIDE TAU
CN113430230A (zh) * 2021-06-24 2021-09-24 南通大学 tau截断体蛋白在诱导tau病理聚集中的应用
CN113490505A (zh) * 2018-11-19 2021-10-08 德克萨斯大学系统董事会 用于治疗tau蛋白病的tau肽抗原及与其结合的抗体
WO2021236809A2 (en) * 2020-05-19 2021-11-25 Othair Prothena Limited Multi-epitope vaccine for the treatment of alzheimer's disease

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104185640A (zh) * 2011-09-19 2014-12-03 阿克松神经系统科学公司 阿尔茨海默病中的tau蛋白介导病变的基于蛋白质的疗法和诊断
WO2019084488A1 (en) * 2017-10-27 2019-05-02 United Neuroscience IMMUNOGENIC CONSTRUCTS OF PEPTIDE TAU
CN113490505A (zh) * 2018-11-19 2021-10-08 德克萨斯大学系统董事会 用于治疗tau蛋白病的tau肽抗原及与其结合的抗体
WO2021236809A2 (en) * 2020-05-19 2021-11-25 Othair Prothena Limited Multi-epitope vaccine for the treatment of alzheimer's disease
CN113430230A (zh) * 2021-06-24 2021-09-24 南通大学 tau截断体蛋白在诱导tau病理聚集中的应用

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