WO2017152399A1 - 一种血管活性肠肽的融合蛋白及其制备方法和应用 - Google Patents

一种血管活性肠肽的融合蛋白及其制备方法和应用 Download PDF

Info

Publication number
WO2017152399A1
WO2017152399A1 PCT/CN2016/075992 CN2016075992W WO2017152399A1 WO 2017152399 A1 WO2017152399 A1 WO 2017152399A1 CN 2016075992 W CN2016075992 W CN 2016075992W WO 2017152399 A1 WO2017152399 A1 WO 2017152399A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
amino acid
acid sequence
vip
vasoactive intestinal
Prior art date
Application number
PCT/CN2016/075992
Other languages
English (en)
French (fr)
Inventor
李红玉
李加忠
朱红梅
支德娟
李洋
高江利
张丽芸
王亚军
Original Assignee
兰州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 兰州大学 filed Critical 兰州大学
Priority to PCT/CN2016/075992 priority Critical patent/WO2017152399A1/zh
Publication of WO2017152399A1 publication Critical patent/WO2017152399A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention belongs to the field of genetic engineering pharmacy, and particularly relates to a fusion protein of vasoactive intestinal peptide and a preparation method and application thereof.
  • Vasoactive intestinal peptide is a 28 amino acid polypeptide produced mainly by the peripheral and central nervous systems, released by the parasympathetic ganglia fibers, and coexisted with acetylcholine. A member of the glucagon-trypticoid family. Widely distributed in the choline-like presynaptic nerve cells and peripheral peptide-energy nerve cells of the central nervous system, through which various organs can be innervated, such as the heart, lungs, digestive system and genitourinary tract, eyes, skin, Ovary and thyroid gland.
  • VIP is an important signal peptide in the central and peripheral nervous systems. It plays a physiological role through its receptor. To date, three types of VIP receptors have been cloned and classified, namely VPAC1, VPAC2 and PAC1. Vasoactive intestinal peptides are involved in a variety of biological functions in the body, including participation in metabolism, exocrine and endocrine, cell differentiation and smooth muscle relaxation, hormone secretion, and regulation of immune responses. Based on its many biological functions, VIP is considered to be a drug candidate with therapeutic potential for a variety of diseases including diabetes, asthma, impotence and rheumatism.
  • VIP has a wide range of biological activities, indicating that VIP has broad clinical application prospects. However, only a few clinical trials have been conducted so far. A major obstacle to the clinical application of VIP is that it is subjected to protease action, antibody neutralization, and spontaneous hydrolysis in vivo with a short half-life, which results in a half-life of VIP in the body of less than one minute. In order to extend the half-life of the VIP, it is necessary to improve its stability by modifying and modifying the structure and other methods.
  • Fusion protein technology using human serum albumin (HSA) as a vector is a method of increasing the half-life of proteins.
  • HSA human serum albumin
  • HSA is the main component of human serum, it plays a vital role in the maintenance of plasma volume and osmotic pressure in vivo, and has no enzymatic activity and immunogenicity, good human compatibility, and high molecular weight (about 66KDa).
  • Very low renal clearance, long half-life, about 19 days, high microbial fermentation expression, etc. often used as a transport carrier for in vivo factors and drugs in the body.
  • Increasing the molecular weight of a polypeptide/protein drug by constructing a fusion protein technology can extend the half-life of the drug.
  • HSA can be used as a carrier of small molecule proteins in the blood, thereby prolonging the half-life of small molecule proteins in the human body.
  • Yeh et al. found that the half-life of the HSA-CD4 fusion protein expressed by Kluyveromyces in the rabbit model as an animal model reached 140 times that of the CD4 monomer.
  • the half-life of the HSA-IFN ⁇ fusion protein expressed by W. cerevisiae was 18-fold longer in cynomolgus than in IFN ⁇ monomer.
  • the design of the fusion protein itself is a process with complicated procedures and numerous influencing factors, and the VIP cannot be realized only by the simple addition of the sequence. It is well known to those skilled in the art for the purpose of stabilizing and efficiently expressing and prolonging their half-life.
  • the inventors have disclosed a fusion protein of vasoactive intestinal peptide, the unique amino acid sequence of the fusion protein can ensure high-level stable expression in the host, while retaining the original function of the VIP, the half-life in vivo Significantly extended.
  • Another object of the present invention is to provide a method for producing a fusion protein of vasoactive intestinal peptide.
  • Another object of the present invention is to provide a recombinant expression vector.
  • Another object of the invention is to provide a host expression system.
  • Another object of the present invention is to provide a use of a fusion protein of a vasoactive intestinal peptide.
  • the fusion protein of vasoactive intestinal peptide of the present invention comprises 1 HSA and 1 VIP.
  • the fusion protein package also contains a linker peptide, and the HSA is linked to the VIP via a linker peptide.
  • the fusion protein is prepared by yeast cell expression, wherein the yeast is Pichia pastoris.
  • the VIP is located at the N-terminus of the fusion protein
  • the HSA is located at the C-terminus of the fusion protein
  • the fusion protein is represented by the structural formula as VIP-L1-HSA, wherein L1 represents a linker peptide, and the DNA sequence of L1 is GGCGGTGGCGGCAGCGGTGGCGGC, the amino acid sequence is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly.
  • the VIP has the amino acid sequence shown in SEQ ID NO: 2, the DNA sequence encoding the amino acid sequence of the VIP is as shown in SEQ ID NO: 1; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • the HSA has the amino acid sequence shown in SEQ ID NO: 4, and the DNA sequence encoding the amino acid sequence of the HSA is shown in SEQ ID NO: 3; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • amino acid sequence of the fusion protein is set forth in SEQ ID NO: 6, and the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 5.
  • preparation method of the fusion protein of the vasoactive intestinal peptide comprises the following steps:
  • step 3 The recombinant expression vector described in step 3 is transformed into competent E. coli TOP10, and then transformed into a host expression system for expression, that is, the fusion protein is obtained.
  • the host expression system of step 4 is yeast, and the yeast is Pichia methanolica.
  • the VIP is located at the C-terminus of the fusion protein
  • the HSA is located at the N-terminus of the fusion protein
  • the fusion protein is represented by the structural formula as HSA-L2-VIP, wherein L2 represents a linker peptide, the DNA sequence of L2 is GGTGGTGGCGGCAGC, and the amino acid sequence is Gly- Gly-Gly-Gly-Ser.
  • the VIP has the amino acid sequence shown in SEQ ID NO: 8, and the DNA sequence encoding the amino acid sequence of the VIP is shown in SEQ ID NO: 7; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • the HSA has the amino acid sequence shown in SEQ ID NO: 10, and the DNA sequence encoding the amino acid sequence of the HSA is as shown in SEQ ID NO: 9; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • amino acid sequence of the fusion protein is set forth in SEQ ID NO: 12, and the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 11.
  • preparation method of the fusion protein of the vasoactive intestinal peptide comprises the following steps:
  • step (2) 3 The recombinant yeast expression vector described in the step (2) 3 is transformed into competent E. coli TOP10, and then transformed into yeast for expression, that is, the fusion protein is obtained.
  • the yeast is P. methanolophilus.
  • a recombinant expression vector comprising a DNA sequence encoding an amino acid sequence of a fusion protein of a vasoactive intestinal peptide.
  • a host expression system comprising the recombinant expression vector described above.
  • a fusion protein of vasoactive intestinal peptide for preparing anti-inflammatory, anti-injury, cerebrovascular diseases and improving sleep quality.
  • a fusion protein of vasoactive intestinal peptide for use in the preparation of a medicament for treating asthma.
  • the unique amino acid sequence of the fusion protein of the invention can ensure high-level stable expression in the host body, and the half-life of the body is significantly prolonged while retaining the original function of the VIP.
  • yeast extract 5 g of yeast extract, 10 g of peptone, 10 g of NaCl, dissolved in 1000 ml of deionized water, and adjusted to pH 7.0 with 1 mol/L NaOH, autoclaved.
  • Yeast extract 10g peptone 20g, amino acid yeast nitrogen source 13.4g, glycerol 10g, potassium phosphate 26.631g, autoclaved in 1000ml double distilled water, cooled to room temperature, adjusted to pH 6.0, stored at 4 ° C for use.
  • agarose a small amount of ethidium bromide (EB) when it is not hot. Mix it and pour it into it. Put the comb in the glue tank beforehand, cool it until it is completely solidified, and then pull out the comb to use.
  • EB ethidium bromide
  • L29 which is a linker peptide
  • the L1 DNA sequence is GGCGGTGGCGGCAGCGGTGGCGGC
  • the amino acid sequence is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly.
  • PCR was carried out using the DNA of the vector pcDNA3.1-HSA as a template and P1 and P2 as the upstream and downstream primers, respectively.
  • the reaction conditions were as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return step "2", 35 cycles; 72 ° C, 5 min, the total number of cycles is 30 times.
  • the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a DNA band of HSA of about 1.8 kb in size was amplified.
  • the vector p29-simple-VIP, StuI and BspEI were digested, and the corresponding VIP (StuI/BspEI) DNA fragment was recovered.
  • the DNA sequence was as shown in SEQ ID NO: 1, and the amino acid sequence was SEQ ID NO: 2. Shown
  • HSA was digested with PCR-amplified products KpnI and BspEI, and the corresponding HSA (KpnI/BspEI) DNA fragment was recovered by gel, the DNA sequence is shown in SEQ ID NO: 3, and the amino acid sequence is SEQ ID NO: 4. Shown
  • T4 DNA enzyme ligated the VIP (StuI/BspEI) DNA fragment, HSA (KpnI/BspEI) DNA fragment and pPink ⁇ -HC (KpnI/StuI) vector fragment, transformed competent E. coli TOP10, and coated on ampicillin-resistant LB plate 37 Incubate overnight at °C and screen positive clones. The resulting clone was sequenced by Invitrogen, and the clone with the correct sequence was named pPink ⁇ -HC/VIP-L1-HSA.
  • the DNA of the correctly sequenced vector pPink ⁇ -HC/VIP-L1-HSA was digested with AflII to obtain pPink ⁇ -HC/VIP-L1-HSA, which was transformed into yeast competent cells.
  • the transformed bacterial solution was then inoculated on a PAD plate, cultured at 30 ° C for 3-4 days, and positive clones were picked.
  • the positive clones were inoculated into BMGY liquid medium, cultured at 30 ° C for 48 hours, then transferred to BMMY medium to induce expression. After 96 hours, centrifuge at 1500 rpm for 15 minutes, the supernatant was taken, and protein expression was detected by SDS-PAGE electrophoresis.
  • the molecular band of about 70 kD protein is the VIP-L1-HSA fusion protein
  • the amino acid sequence of the fusion protein is SEQ ID NO: 6
  • the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 5. Show. Select the strain with the highest expression level as the engineering strain, and store it at -80 °C for preservation.
  • p29-simple-VIP already contains L2, which is a linker peptide, the L2 DNA sequence is GGTGGTGGCGGCAGC, and the amino acid sequence is Gly-Gly-Gly-Gly-Ser.
  • PCR was carried out using the DNA of the vector pcDNA3.1-HSA as a template and P1 and P2 as the upstream and downstream primers, respectively.
  • the reaction conditions were as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return step "2", 35 cycles; 72 ° C, 5 min, the total number of cycles is 30 times.
  • the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a DNA band of HSA of about 1.8 kb in size was amplified.
  • HSA was digested with PCR-amplified products EcoRI and HindIII, and the corresponding HSA (EcoRI/HindIII) DNA fragment was recovered by gel.
  • the DNA sequence is shown in SEQ ID NO: 9, and the amino acid sequence is as shown in SEQ ID NO: 10.
  • 4T4 DNase was ligated with VIP (EcoRI/XhoI) DNA fragment, HSA (EcoRI/HindIII) DNA fragment and pcDNA3.1 (XhoI/HindIII) vector fragment, transformed into competent E. coli TOP10, and applied to ampicillin-resistant LB plate at 37 ° C overnight. , screening positive clones. The resulting clone was sequenced by Invitrogen, and the clone with the correct sequence was named pcDNA3.1-HSA-L2-VIP.
  • the obtained HSA-L2-VIP DNA fragment has a DNA sequence as shown in SEQ ID NO: 11, and an amino acid sequence as shown in SEQ ID NO: 12.
  • the DNA of the pPink ⁇ -HC (KnI/StuI) vector fragment was recovered by double-cleaving the DNA of the vector pPink ⁇ -HC (product of Invitrogen) with 1KpnI and StuI.
  • the DNA of the correctly sequenced vector pPink ⁇ -HC/HSA-L2-VIP was digested with AflII to obtain pPink ⁇ -HC/HSA-L2-VIP, transformed into yeast competent cells.
  • the transformed bacterial solution was then inoculated on a PAD plate, cultured at 30 ° C for 3-4 days, and clones were picked.
  • the positive clones were inoculated into BMGY liquid medium, cultured at 30 ° C for 48 hours, then transferred to BMMY medium to induce expression. After 96 hours, centrifuge at 1500 rpm for 15 minutes, the supernatant was taken, and protein expression was detected by SDS-PAGE electrophoresis.
  • the molecular band of about 70 kD protein is the HAS-L2-VIP fusion protein
  • the amino acid sequence of the fusion protein is SEQ ID NO: 12
  • the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: Show.
  • the strain with the highest expression level was selected as the engineering bacteria, and stored at -80 °C for preservation.
  • Example 3 Detection of biological activity of fusion protein of vasoactive intestinal peptide
  • Human colon cancer cell line HT-29 was purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences.
  • a 24-well plate was plated with 2 ⁇ 105 HT-29 cells per well, 1 mL per well. After the cell confluence was 80%, wash once with PBS, add 0.1 mmol/L IBMX, 500 mL per well, and after 30 min of treatment, add The fusion protein and VIP of purified vasoactive intestinal peptide were expressed in Pichia pastoris, 500uL per well. After 30min treatment, the supernatant was aspirated, washed 3 times with cold PBS, finally washed, and then added with appropriate cell lysate. It was frozen in a refrigerator at -20 ° C for 30 min, and slowly thawed at room temperature, and it was allowed to freeze and thaw repeatedly until the cells ruptured. The supernatant was collected by centrifugation at 600 g, 4 ° C, and stored at -20 ° C until use.
  • the biological activity of the above-prepared cell lysate was measured by a competitive ELISA kit: except for the NSB (non-specific binding) well, a primary anti-50 uL was added to each well, and the supernatant was decanted after incubating at 37 ° C for 1 hour. Wash 4 times with Wash Buffer for 1 min each time, then add 50 uL of cAMP Conjugate to each well, add standard solution and corresponding sample in 15 min, 100 uL per well, and incubate at 37 °C for 2 h.
  • NSB non-specific binding
  • the blank control was DMEM/F12
  • the negative control was empty vector (pPink ⁇ -HC) transferred to the supernatant of Pichia methanolica broth
  • the positive control was 1 ⁇ 10 -9 mol/L VIP.
  • the fusion protein of the vasoactive intestinal peptide disclosed by the present invention has higher activity and better anti-inflammatory effect than the positive control group.
  • Thirty wister rats were divided into six groups, about 180-220 g: 1-control group, 2-model group, 3-dexamethasone group, 4-VIP group 1,5-VIP group 2,6-vasoactive intestinal A fusion protein group of peptides.
  • the control group was intraperitoneally injected with 1 mL of normal saline, and the other groups were given 1 mL of physiological saline (containing 2 mg of OVA and 100 mg of aluminum hydroxide) to induce sensitization.
  • the other groups except the control were challenged with 1% OVA once a day for 30 minutes for 7 days; the control group was replaced with physiological saline.
  • the dexamethasone group was intraperitoneally injected with dexamethasone in the first half hour before each nebulization, VIP was injected into VIP group 1 and 2, and the fusion protein of vasoactive intestinal peptide was intraperitoneally injected with vasoactive intestinal peptide.
  • Rats with bronchoalveolar lavage fluid were taken within 24 hours after the last aerosol challenge on the seventh day of dexamethasone group and VIP group.
  • the counts of various inflammatory cells were counted to investigate the changes in cytology after asthma.
  • the blood levels of abdominal aorta were detected by ELISA.
  • the levels of tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin-4 (IL-4) were detected by ELISA.
  • the lung tissue of rats was taken to make pathological specimens of lung tissue to observe the morphology of lung tissue. Variety.
  • VIP group 2 stopped the injection of VIP, and the fusion protein of vasoactive intestinal peptide stopped the injection of vasoactive intestinal peptide fusion protein.
  • the two groups continued to be stimulated with 1% OVA once a day for 30 minutes each time.
  • the rat bronchoalveolar lavage fluid (BALF) was taken within 24 hours after the last atomization challenge.
  • the counts of various inflammatory cells were counted to investigate the changes of cytology after asthma.
  • the abdominal aorta was taken by ELISA.
  • the levels of tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin-4 (IL-4) in serum were changed.
  • the lung tissue of rats was taken to prepare pathological specimens of lung tissue to observe the morphological changes of lung tissue.
  • mice The weight of the normal mice is slower than normal, and it is irritated, sneezing, and tarnished; the breathing is rapid, coughing, forelimb contraction, nodding or abdominal breathing, irregular rhythm, slow movement, asthma The performance, and the symptoms increase with the increase in the number of attacks.
  • the fusion protein group of VIP group 1 and vasoactive intestinal peptide was similar to or similar to the control group.
  • the levels of tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin-4 (IL-4) in serum were detected by ELISA.
  • the levels of 2IL-4 and TNF- ⁇ in the model group and VIP group were significantly higher than those in the control group.
  • the fusion protein group of VIP group 1 and vasoactive intestinal peptide is similar to or close to the control group.
  • the VIP protein has anti-inflammatory, anti-injury, cerebrovascular diseases, and the effect of improving sleep quality
  • the fusion protein of the vasoactive intestinal peptide disclosed in the present invention has the same efficacy, which can be understood by those skilled in the art.
  • the invention discloses a fusion protein of vasoactive intestinal peptide in preparing anti-inflammatory, anti-injury, cerebrovascular diseases and improving sleep The application of sleep quality drugs.
  • the invention discloses a use of a fusion protein of vasoactive intestinal peptide in the preparation of a medicament for treating asthma.
  • the unique amino acid sequence of the fusion protein disclosed in the invention can ensure high-level stable expression in the host, and the half-life of the body is significantly prolonged while retaining the original function of the VIP.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种血管活性肠肽的融合蛋白，所述融合蛋白包含1个人血清白蛋白（Albumin Human，HAS）和1个血管活性肠肽（vasoactive intestinal peptide，VIP）。该融合蛋白所具有的独特的氨基酸序列可以保证其在宿主体内高水平稳定表达，在保留VIP原有功能的同时，体内半衰期显著延长。同时提供该融合蛋白的制备方法及其在制备抗炎、抗损伤、脑血管疾病、提高睡眠质量的药物中的应用。

Description

一种血管活性肠肽的融合蛋白及其制备方法和应用 技术领域
本发明属于基因工程制药领域，具体涉及一种血管活性肠肽的融合蛋白及其制备方法和应用.
背景技术
血管活性肠肽(vasoactive intestinal peptide，VIP)是一种由28个氨基酸组成的多肽，主要是由外周神经和中枢神经系统产生，由副交感神经节后纤维释放，并且与乙酰胆碱共同存在，被认为是胰高血糖素-胰泌素家族的一员。广泛分布在中央神经系统的类胆碱突触前神经细胞和外周肽能神经细胞中，通过它可对多种器官进行神经支配，比如心脏，肺，消化系统和泌尿生殖道，眼睛，皮肤，卵巢和甲状腺。
VIP在中枢和外周神经系统中是一种重要的信号肽，通过其受体发挥生理作用，迄今为止，已有3种类型的VIP受体被克隆和分类，分别为VPAC1、VPAC2和PAC1。血管活性肠肽在生物体内涉及到多种生物学功能，包括参与新陈代谢、外分泌和内分泌、细胞分化和平滑肌舒张、激素分泌和免疫应答调控。基于它的多种生物学功能，VIP被认为是一种对多种疾病包括糖尿病、哮喘，阳痿和风湿有治疗前景的药物候选物。
VIP具有广泛的生物学活性，表明VIP具有广阔的临床应用前景。但是，到目前为止只开展了少数临床实验。VIP临床应用的一个主要障碍就是它在体内受到蛋白酶作用、抗体中和、自发水解而半衰期很短，这使VIP在体内半衰期不足1分钟。为了延长VIP的半衰期，就要通过对其进行结构的修饰和改造以及其他方法来提高其稳定性。
尝试提高VIP的稳定性的方法，包括对其母体进行化学修饰、使用稳定的VIP受体激动剂或VIP类似物、VIP与肽酶抑制剂组合使用或将VIP准确地嵌入到微粒或纳米粒子中。氨基酸的化学修饰或替换作为一种常用的方法早已被提出并用来增加神经肽的稳定性。Onoue和他的同事得到了两个VIP的衍生物具有相当可观的稳定性(在40℃超过60天)，它被做成可吸入的粉末制剂在哮喘和慢性阻塞性肺病(COPD)模型上具有一定功效。但是这些VIP的衍生物在治疗其他疾病上面受到了限制。之前也有提出使用微胶粒或脂质体包埋神经肽使其在体内缓慢释放，近期这种方法也被用于VIP的体内抗炎模型的应用上。然而这种脂质体包埋的神经肽进入目标器官受到了其颗粒大小的限制。为了克服这个潜在的问题，一个研究描述了一种将VIP包埋于由银保护的纳米粒子，它的功能和体内固定VIP的小神经胶质细胞相类似，这项研究第一次提供了使用金属纳米粒子对神经肽靶向给药的资料，但是在炎症状态下的基于纳米粒子的治疗效果还需进一步证明。虽然已有多种延长VIP在体内半衰期的方法，但是 仍需找到一种简单、经济且安全可靠的方法。
以人血清白蛋白(Albumin Human，HSA)为载体的融合蛋白技术是一个受关注的延长蛋白质半衰期的方法。由于HSA是人血清的主要成分，对体内血浆体积和渗透压的维持起到了至关重要的作用，而且无酶活性和免疫原性，人体相容性好，分子量大(约为66KDa),具有非常低的肾清除率，半衰期长，约为19天，微生物发酵表达量高等优点，在体内常作为体内因子和药物的转运载体。通过构建融合蛋白技术增加多肽/蛋白类药物分子量可延长药物半衰期。所以，HSA可以作为小分子蛋白在血液中的载体，从而延长小分子蛋白在人体内的半衰期。Yeh等的研究发现，通过克鲁维氏酵母表达的HSA-CD4融合蛋白在以家兔作为动物模型的实验中，其半衰期达到了CD4单体的140倍。通过维氏酵母表达的HSA-IFNα融合蛋白，它的半衰期在猕猴体内比IFNα单体延长了18倍。
虽然现有技术中公开了通过融合蛋白的表达的策略可以延长目的蛋白的半衰期，但是融合蛋白的设计本身是一个程序复杂、影响因素众多的过程，仅通过序列的简单加和是无法实现VIP的稳定高效表达、延长其半衰期的目的的，这是本领域技术人员所公知的。
基于上述现状，发明人公开了一种血管活性肠肽的融合蛋白，该融合蛋白所具有的独特的氨基酸序列可以保证其在宿主体内高水平稳定表达，在保留VIP原有功能的同时，体内半衰期显著延长。
发明内容
本发明的目的在于提供一种血管活性肠肽的融合蛋白，该融合蛋白半衰期长，能在宿主体内高水平稳定表达。
本发明的另一个目的在于提供一种血管活性肠肽的融合蛋白的制备方法。
本发明的另一个目的在于提供一种重组表达载体。
本发明的另一个目的在于提供一种宿主表达系统。
本发明的另一个目的在于提供一种血管活性肠肽的融合蛋白的应用。
本发明所述的血管活性肠肽的融合蛋白，包含1个HSA和1个VIP。
所述融合蛋白包还含有一个连接肽，HSA通过连接肽与VIP连接。
所述融合蛋白采用酵母细胞表达制备，其中，酵母为嗜甲醇毕赤酵母(Pichia pastoris)。
(一)VIP-L1-HSA
所述VIP位于融合蛋白的N-末端，HSA位于融合蛋白的C-末端，融合蛋白用结构式表示为VIP-L1-HSA，其中L1表示连接肽，L1的DNA序列为 GGCGGTGGCGGCAGCGGTGGCGGC，氨基酸序列为Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly。
其中，所述VIP具有SEQ ID NO：2所示的氨基酸序列，编码所述VIP的氨基酸序列的DNA序列如SEQ ID NO：1所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述VIP的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
所述HSA具有SEQ ID NO：4所示的氨基酸序列，编码所述HSA的氨基酸序列的DNA序列如SEQ ID NO：3所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述人血清白蛋白HSA的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
所述融合蛋白的氨基酸序列如SEQ ID NO：6所示，编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO：5所示。
其中，所述的血管活性肠肽的融合蛋白的制备方法包含以下步骤：
①全基因合成VIP序列；
②通过PCR扩增获取HSA序列；
③通过限制性内切酶酶切、连接并转化大肠杆菌，获得含编码所述血管活性肠肽的融合蛋白的氨基酸序列的DNA序列的重组表达载体；
④将步骤③所述的重组表达载体转化到感受态大肠杆菌TOP10，再转化到宿主表达系统进行表达，即得所述融合蛋白。
步骤④所述宿主表达系统是酵母，所述的酵母为嗜甲醇毕赤酵母。
(二)HSA-L2-VIP
所述VIP位于融合蛋白的C-末端，HSA位于融合蛋白的N-末端，融合蛋白用结构式表示为HSA-L2-VIP，其中L2表示连接肽，L2的DNA序列为GGTGGTGGCGGCAGC，氨基酸序列为Gly-Gly-Gly-Gly-Ser。
所述VIP具有SEQ ID NO：8所示的氨基酸序列，编码所述VIP的氨基酸序列的DNA序列如SEQ ID NO：7所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述VIP的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
所述HSA具有SEQ ID NO：10所示的氨基酸序列，编码所述HSA的氨基酸序列的DNA序列如SEQ ID NO：9所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述HSA的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
所述融合蛋白的氨基酸序列如SEQ ID NO：12所示，编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO：11所示。
其中，所述的血管活性肠肽的融合蛋白的制备方法包含以下步骤：
(1)所述血管活性肠肽的融合蛋白的动物表达载体的构建
①全基因合成VIP序列；
②通过PCR扩增获取HSA序列；
③通过限制性内切酶酶切、连接并转化大肠杆菌，获得含编码所述血管活性肠肽的融合蛋白的氨基酸序列的DNA序列的重组动物表达载体；
(2)所述血管活性肠肽的融合蛋白的酵母表达载体的构建
①通过PCR扩增步骤(1)③中重组动物表达载体获取HSA-L2-VIP；
②通过限制性内切酶酶切酵母表达载体；
③使用融合PCR技术连接(2)①中HSA-L2-VIP与(2)②中酵母表达载体，并转化大肠杆菌，获得含编码所述血管活性肠肽的融合蛋白的氨基酸序列的DNA序列的重组酵母表达载体；
(3)将步骤(2)③所述的重组酵母表达载体转化到感受态大肠杆菌TOP10，再转化到酵母中进行表达，即得所述融合蛋白。
所述的酵母为嗜甲醇毕赤酵母。
一种含有编码血管活性肠肽的融合蛋白的氨基酸序列的DNA序列的重组表达载体。
一种含有上述的重组表达载体的宿主表达系统。
一种血管活性肠肽的融合蛋白在制备抗炎、抗损伤、脑血管疾病、提高睡眠质量的药物中的应用。
一种血管活性肠肽的融合蛋白在制备治疗哮喘药物中的应用。
本发明所述融合蛋白所具有的独特的氨基酸序列可以保证其在宿主体内高水平稳定表达，在保留VIP原有功能的同时，体内半衰期显著延长。
附图说明
图1载体pPinkα-HC
图2载体pPinkα-HC/VIP-L1-HSA
图3载体pcDNA3.1
图4载体pcDNA3.1-HSA
图5载体pcDNA3.1-HSA-L2-VIP
图6载体pPinkα-HC/HSA-L2-VIP
具体实施方式
主要实验仪器
移液枪、超净工作台(安泰)、磁力搅拌器、微波炉、高温蒸汽灭菌锅、-80℃低温冰箱(Forma)、超纯水仪(Millipore)、制冰机、离心机(Hitachi)、HDB-PLUS型恒温金属浴、HZQ-F16OA型恒温振荡培养箱(上海一恒)、PCR仪(Applied Biosystems)、台式冷冻离心机(Thermo)、DYY-8B型电泳仪(伯乐)、Image Quant 300型凝胶成像仪(GE)等。
主要实验材料：
1.限制性核酸内切酶StuI、BspEI、KpnI、XbaI、EcoRI、XhoI、HindⅢ、AflII(NEB公司产品，美国)
2.小提质粒试剂盒、PCR纯化试剂盒、DNA胶回收试剂盒(生工公司，中国)
3.T4DNA连接酶试剂盒(Takara公司产品，中国大连)
4.载体pPinkα-HC、载体pcDNA3.1-HSA、嗜甲醇毕赤酵母菌株(Invitrogen公司产品，美国)
5.大肠杆菌TOP10(天根生化科技(北京)有限公司)
6.酵母提取物、蛋白胨(Oxford公司产品，美国)
7.LB培养基
酵母提取物5g，蛋白胨10g，NaCl 10g，溶于1000ml去离子水中，并用1mol/L的NaOH调节pH值至7.0，高压蒸气灭菌。
8.YPD培养基
酵母提取物10g，胰蛋白胨20g，Agar 20g，溶于900ml去离子水中，高压灭菌，冷却后加入100ml经滤器除菌后的20％的右旋糖。
9.YPDS培养基
酵母提取物10g，蛋白胨20g，山梨糖醇182.2g，溶于900ml去离子水中，高压灭菌，冷却后加入100ml经滤器除菌后的20％的右旋糖。
10.BMGY液体培养基
酵母提取物10g，蛋白胨20g，无氨基酸酵母氮源13.4g，甘油10g，磷酸钾26.631g，溶于1000ml双蒸水中高压灭菌，冷至室温，调节pH至6.0，4℃保存备用。
11. 1％琼脂糖凝胶的配置
根据用量，每100ml的TAE缓冲液，加入1g琼脂糖，使用微波炉加热煮沸，使琼脂糖完全融解，室温冷却至不烫手时滴加少量溴化乙锭(EB)，混匀后将其倒入事先摆放好梳子的胶槽中，待到室温冷却至完全凝固后拔去梳子即可使用。
实施例1  VIP-L1-HSA融合蛋白酵母表达载体的构建与表达
一、p29-simple-VIP序列的获得
1.委托大连Takara公司合成优化后的VIP基因，VIP的DNA序列如SEQ ID NO：1所示，并将其装载到p29-simple(p29-simple质粒载体大连Takara公司提供)，获得载体p29-simple-VIP。
2.其中，p29-simple-VIP中已包含L1，即连接肽，L1DNA序列为GGCGGTGGCGGCAGCGGTGGCGGC，氨基酸序列为Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly。
二、HSA序列的获得
1.设计合成PCR引物：
P1:GGTACCTCATAAGCCTAAGGCAGCTTG
P2:TCCGGAGATGCACACAAGAGTGAG
2.PCR扩增：以载体pcDNA3.1-HSA的DNA为模版，以P1和P2分别作为上、下游引物，进行PCR扩增。反应条件如下：①变性：94℃，5min；②变性：94℃，1min；③复性：55℃，30S；④延伸：72℃，2min；⑤返回步骤“②”，35循环；⑥延伸：72℃，5min，总循环次数为30次。将PCR产物进行1％琼脂糖凝胶电泳，结果显示扩增出约1.8kb大小的HSA的DNA条带。
三、VIP-L1-HSA融合蛋白酵母表达载体的构建
1.提取载体p29-simple-VIP，StuI和BspEI双酶切质粒,胶回收对应的VIP(StuI/BspEI)DNA片段,DNA序列如SEQ ID NO：1所示，氨基酸序列如SEQ ID NO：2所示；
2.将HSA经PCR扩增后的产物KpnI和BspEI双酶切，胶回收对应的HSA(KpnI/BspEI)DNA片段,DNA序列如SEQ ID NO：3所示，氨基酸序列如SEQ ID NO：4所示；
3.同时，KpnI和StuI双酶切载体pPinkα-HC(Invitrogen公司产品)的DNA，胶回收pPinkα-HC(KpnI/StuI)载体片段。
4.T4DNA酶连接VIP(StuI/BspEI)DNA片段、HSA(KpnI/BspEI)DNA片段和pPinkα-HC(KpnI/StuI)载体片段，转化感受态大肠杆菌TOP10，涂布于氨苄抗性LB板37℃培养过夜，筛选阳性克隆。所得克隆送Invitrogen公司测序，序列正确的克隆命名为pPinkα-HC/VIP-L1-HSA。
四、VIP-L1-HSA融合蛋白在酵母中的表达
将测序正确的载体pPinkα-HC/VIP-L1-HSA的DNA用AflII酶切回收后得到pPinkα-HC/VIP-L1-HSA，转化酵母感受态细胞。然后将转化菌液接种于PAD平板，30℃培养3-4天，挑取阳性克隆。将得到阳性克隆分别接种BMGY液体培养基，30℃培养48小时，然后转接至BMMY培养基中诱导表达，持续96小时后，1500rpm低温离心15分钟，取上清，SDS-PAGE电泳检测蛋白表达情况，分子量约70kD蛋白条带即为VIP-L1-HSA融合蛋白，所述融合蛋白的氨基酸序列如SEQ ID NO：6，编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO：5所示。选择表达水平最高的菌株作为工程菌，冻存于-80℃保种
实施例2  HSA-L2-VIP融合蛋白酵母表达载体的构建与表达
一、HSA-L2-VIP融合蛋白动物表达载体的构建
1.p29-simple-VIP序列的获得
①.委托大连Takara公司合成优化后的VIP基因，VIP的DNA序列如SEQ ID NO：7所示，并将其装载到p29-simple(p29-simple质粒载体大连Takara公司提供)，获得载体p29-simple-VIP。
②.其中，p29-simple-VIP中已包含L2，即连接肽，L2DNA序列为GGTGGTGGCGGCAGC，氨基酸序列为Gly-Gly-Gly-Gly-Ser。
2.HSA序列的获得
①设计合成PCR引物：
P1:GAAGAAGCTTTGCTATGGAGACAGACACACTCCTG
P2:GAAGGAATTCGTGGTGGTGGTGGTGGTGGCAGGGAGGGCAGGTGTGGGTCTTG
②PCR扩增：以载体pcDNA3.1-HSA的DNA为模版，以P1和P2分别作为上、下游引物，进行PCR扩增。反应条件如下：①变性：94℃，5min；②变性：94℃，1min；③复性：55℃，30S；④延伸：72℃，2min；⑤返回步骤“②”，35循环；⑥延伸：72℃，5min，总循环次数为30次。将PCR产物进行1％琼脂糖凝胶电泳，结果显示扩增出约1.8kb大小的HSA的DNA条带。
3.HSA-L2-VIP融合蛋白动物表达载体的构建
①提取载体p29-simple-VIP，EcoRI和XhoI双酶切质粒,胶回收对应的VIP(EcoRI/XhoI)DNA片段,DNA序列如SEQ ID NO：7所示，氨基酸序列如SEQ ID NO：8所示；
②将HSA经PCR扩增后的产物EcoRI和HindⅢ双酶切，胶回收对应的HSA(EcoRI/HindⅢ)DNA片段,DNA序列如SEQ ID NO：9所示，氨基酸序列如SEQ ID NO：10所示；
③同时，XhoI和HindⅢ双酶切pcDNA3.1质粒DNA，胶回收pcDNA3.1(XhoI/HindⅢ)载体片段。
④T4DNA酶连接VIP(EcoRI/XhoI)DNA片段、HSA(EcoRI/HindⅢ)DNA片段和pcDNA3.1(XhoI/HindⅢ)载体片段，转化感受态大肠杆菌TOP10，涂于氨苄抗性LB板37℃培养过夜，筛选阳性克隆。所得克隆送Invitrogen公司测序，序列正确的克隆命名为pcDNA3.1-HSA-L2-VIP。
二、HSA-L2-VIP融合蛋白酵母表达载体的构建
1.HSA-L2-VIP序列的获得
①设计合成PCR引物：
P1:TCTCTCGAGAAAAGGGATGCACACAAGAGTGAGGTTGC
P2:TTAAATGGCCGGCCGGTACCTCAATTCAGAATTGAGTTC
②PCR扩增：以载体pcDNA3.1-HSA-VIP的DNA为模版，以P1和P2分别作为上、下游引物，进行PCR扩增得到。反应条件如下：①变性：94℃，5min；②变性：94℃，1min；③复性：55℃，30S；④延伸：72℃，2min；⑤返回步骤“②”，35循环；⑥延伸：72℃，5min，总循环次数为30次。得到的HSA-L2-VIP DNA片段,DNA序列如SEQ ID NO：11所示，氨基酸序列如SEQ ID NO：12所示；
2.HSA-VIP融合蛋白酵母表达载体的构建
①KpnI和StuI双酶切载体pPinkα-HC(Invitrogen公司产品)的DNA，胶回收pPinkα-HC(KpnI/StuI)载体片段。
②使用融合PCR技术将上述pPinkα-HC(KpnI/StuI)载体片段和HSA-L2-VIP DNA片段连接，转化感受态大肠杆菌TOP10，涂布于氨苄抗性LB板37℃培养过夜，筛选阳性克隆。所得克隆送Invitrogen公司测序，序列正确的克隆命名为pPinkα-HC/HSA-L2-VIP。
三、HSA-L2-VIP融合蛋白在酵母中的表达
将测序正确的载体pPinkα-HC/HSA-L2-VIP的DNA用AflII酶切回收后得到 pPinkα-HC/HSA-L2-VIP，转化酵母感受态细胞。然后将转化菌液接种于PAD平板，30℃培养3-4天，挑取克隆。将得到阳性克隆分别接种BMGY液体培养基，30℃培养48小时，然后转接至BMMY培养基中诱导表达，持续96小时后，1500rpm低温离心15分钟，取上清，SDS-PAGE电泳检测蛋白表达情况，分子量约70kD蛋白条带即为HAS-L2-VIP融合蛋白，所述融合蛋白的氨基酸序列如SEQ ID NO：12，编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO：11所示。选择表达水平最高的菌株作为工程菌，冻存于-80℃保种。
实施例3：血管活性肠肽的融合蛋白生物活性检测
实验材料
人结肠癌细胞HT-29，购自中国医学科学院基础医学研究所基础医学细胞中心。
ELISA试剂盒，购自美国R&D公司。
(1)制备细胞裂解液
以每孔2×105个HT-29细胞进行24孔板铺板，每孔1mL,待细胞汇合度为80％时用PBS洗涤一次后加0.1mmol/L IBMX，每孔500mL,处理30min后，加入嗜甲醇毕赤酵母表达纯化的血管活性肠肽的融合蛋白和VIP，每孔500uL，处理30min后吸掉上清，用冷的PBS洗涤3次，最后洗干净，再加入适当的细胞裂解液放在-20℃的冰箱中冷冻30min，放在室温下慢慢融化，可反复冻融，直至细胞破裂。600g，4℃离心收集上清，-20摄氏度保存待用。
(2)竞争ELISA试剂盒检测融合蛋白的生物活性
用竞争ELISA试剂盒检测上述制备的细胞裂解液的生物活性：除NSB(非特异结合)孔外，每孔加入初抗50uL,37℃孵化1小时后倒掉上清。用Wash Buffer洗涤4次，每次1min，然后每孔加入50uL的cAMP Conjugate，在15min内加入标准液和相应的样品，每孔100uL，在37℃孵化2h。再用Wash Buffer洗涤4次后，加入substrate solution进行显色，每孔200uL,在37℃孵化30min后，每孔加入100uL stop solution终止显色，在酶标仪上测450nm和540nm处的吸光值，计算cAMP浓度。
实验结果如表1所示：
表1 血管活性肠肽的融合蛋白生物活性检测结果
  空白对照 阴性对照 阳性对照 VIP-L1-HSA HSA-L2-VIP
cAMP(pmol/mL) 12.7635 12.4238 22.6252 30.9549 37.6488
其中，空白对照为DMEM/F12，阴性对照为空载体(pPinkα-HC)转入嗜甲醇毕赤酵母发酵液的上清，阳性对照为1×10-9mol/L的VIP。
其中，cAMP的含量越高，表示供试样品的活性越高，抗炎效果越好。从上表可以看出，本发明公开的血管活性肠肽的融合蛋白较阳性对照组相比，具有更高的活性，抗炎效果更好。
实施例4  血管活性肠肽的融合蛋白的功能验证
实验材料
1.实验仪器
注射器、移液枪、金属浴、离心机(Hitachi)、超纯水仪(Millipore)、涡旋仪、恒温培
养箱(上海一恒)、酶标仪(Thermo)等。
2.实验动物
30只wister大鼠，购于兰州大学实验动物中心。
实验方法
大鼠的分组及给药方式：
将30只wister大鼠分为六组,180～220g左右：1-对照组，2-模型组，3-地塞米松组，4-VIP组1，5-VIP组2，6-血管活性肠肽的融合蛋白组。
第一天和第八天对照组予1mL生理盐水腹腔注射，其他各组给予1mL生理盐水(含OVA2mg、氢氧化铝100mg)以致敏。第十五天除对照外的其余各组以1％OVA进行激发，每天一次，每次30min，连续7天；对照组以生理盐水代替。每次雾化激发前半小时地塞米松组腹腔注射地塞米松，VIP组1、2腹腔注射VIP，血管活性肠肽的融合蛋白组腹腔注射血管活性肠肽的融合蛋白。
地塞米松组和VIP组1第七天最后一次雾化激发后24小时内取大鼠支气管肺泡灌洗液(BALF)，计数其中多种炎性细胞的计数来考察哮喘后细胞学的变化；腹主动脉取血用ELISA法检测血清中肿瘤坏死因子-α(TNF-α)和白介素-4(IL-4)的水平变化；取大鼠肺部组织制作肺组织病理标本观察肺组织形态学变化。
从第八天开始，VIP组2停止注射VIP，血管活性肠肽的融合蛋白组停止注射血管活性肠肽的融合蛋白，两组继续以1％OVA进行激发，每天一次，每次30min，连续3天，最后一次雾化激发后24小时内取大鼠支气管肺泡灌洗液(BALF)，计数其中多种炎性细胞的计数来考察哮喘后细胞学的变化；腹主动脉取血用ELISA法检测血清中肿瘤坏死因子-α(TNF-α)和白介素-4(IL-4)的水平变化；取大鼠肺部组织制作肺组织病理标本观察肺组织形态学变化。
实验结果与分析：
表2 血管活性肠肽的融合蛋白的功效
Figure PCTCN2016075992-appb-000001
a：各项行为正常。
b：较正常鼠体重增长缓慢，并表现为烦躁、打喷嚏、毛色失去光泽；雾化激发时呼吸急促、呛咳、前肢缩抬、点头或腹式呼吸，节律不规则，行动迟缓，呈哮喘样表现，并随着激发次数增加症状逐步加重。
c：未见气道炎症改变，肺内支气管管壁无增厚、支气管管腔规则、粘膜上皮整齐，管腔内无炎性渗出物，气道周围未见明显炎性细胞浸润。
d：支气管周围可见炎症细胞浸润，支气管内可见粘液栓；气道上皮有脱落、断裂。
由表2中的结果可以看出，对照组大鼠行为正常，肺组织病理学切片HE染色观察未见气道炎症改变，肺内支气管管壁无增厚、支气管管腔规则、粘膜上皮整齐，管腔内无炎性渗出物，气道周围未见明显炎性细胞浸润。模型组和VIP组2较正常鼠体重增长缓慢，并表现为烦躁、打喷嚏、毛色失去光泽；雾化激发时呼吸急促、呛咳、前肢缩抬、点头或腹式呼吸，节律不规则，行动迟缓，呈哮喘样表现，并随着激发次数增加症状逐步加重。肺组织病理学切片HE染色观察支气管周围可见炎症细胞浸润，支气管内可见粘液栓；气道上皮有脱落、断裂。VIP组1、血管活性肠肽的融合蛋白组与对照组相似或接近。ELISA检测血清中肿瘤坏死因子-α(TNF-α)和白介素-4(IL-4)的水平变化，其中模型组和VIP组2IL-4，TNF-α水平较对照组有明显升高。VIP组1、血管活性肠肽的融合蛋白组与对照组相似或接近
以上实验说明本发明提供的血管活性肠肽的融合蛋白与VIP相比具有较长的半衰期，药效维持时间更长。
文献报道，VIP蛋白具有抗炎、抗损伤、脑血管疾病、提高睡眠质量的功效，本发明公开的血管活性肠肽的融合蛋白具有相同的功效，这是本领域技术人员所能理解的。
本发明公开了一种血管活性肠肽的融合蛋白在制备抗炎、抗损伤、脑血管疾病、提高睡 眠质量的药物中的应用。
本发明公开了一种血管活性肠肽的融合蛋白在制备治疗哮喘的药物中的应用。
本发明公布的融合蛋白所具有的独特的氨基酸序列可以保证其可以在宿主体内高水平稳定表达，在保留VIP原有功能的同时，体内半衰期显著延长。

Claims (21)

  1. 一种血管活性肠肽的融合蛋白，其特征在于，所述融合蛋白包含1个人血清白蛋白(Albumin Human，HSA)和1个血管活性肠肽(vasoactive intestinal peptide，VIP)。
  2. 根据权利要求1所述的一种血管活性肠肽的融合蛋白，其特征在于，所述融合蛋白包还含有一个连接肽，HSA通过连接肽与VIP连接。
  3. 根据权利要求2所述的一种血管活性肠肽的融合蛋白，其特征在于，所述VIP位于融合蛋白的N-末端，HSA位于融合蛋白的C-末端，融合蛋白用结构式表示为VIP-L1-HSA，其中L1表示连接肽，L1的DNA序列为GGCGGTGGCGGCAGCGGTGGCGGC，氨基酸序列为Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly。
  4. 根据权利要求3所述的一种血管活性肠肽的融合蛋白，其特征在于，所述VIP具有SEQ ID NO：2所示的氨基酸序列，编码所述VIP的氨基酸序列的DNA序列如SEQ ID NO：1所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述VIP的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
  5. 根据权利要求3所述的一种血管活性肠肽的融合蛋白，其特征在于，所述HSA具有SEQ ID NO：4所示的氨基酸序列，编码所述HSA的氨基酸序列的DNA序列如SEQ ID NO：3所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述HSA的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
  6. 根据权利要求1-3中任意一项所述的一种血管活性肠肽的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO：6所示，编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO：5所示。
  7. 根据权利要求2所述的一种血管活性肠肽的融合蛋白，其特征在于，所述VIP位于融合蛋白的C-末端，HSA位于融合蛋白的N-末端，融合蛋白用结构式表示为HSA-L2-VIP，其中L2表示连接肽，L2的DNA序列为GGTGGTGGCGGCAGC，氨基酸序列为Gly-Gly-Gly-Gly-Ser。
  8. 根据权利要求7所述的一种血管活性肠肽的融合蛋白，其特征在于，所述VIP具有SEQ ID NO：8所示的氨基酸序列，编码所述VIP的氨基酸序列的DNA序列如SEQ ID NO：7所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述VIP的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
  9. 根据权利要求7所述的一种血管活性肠肽的融合蛋白，其特征在于，所述HSA具有SEQ ID NO：10所示的氨基酸序列，编码所述HSA的氨基酸序列的DNA序列如SEQ ID NO：9所示；或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述HSA 的活性的氨基酸序列，及编码所述的氨基酸序列的DNA序列。
  10. 根据权利要求1-2和7中任意一项所述的一种血管活性肠肽的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO：12所示，编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO：11所示。
  11. 根据权利要求1-2所述的一种血管活性肠肽的融合蛋白，其特征在于所述融合蛋白采用酵母细胞表达制备。
  12. 根据权利要求11所述的一种血管活性肠肽的融合蛋白，其特征在于所述的酵母为嗜甲醇毕赤酵母(Pichia pastoris)。
  13. 一种如权利要求3所述的血管活性肠肽的融合蛋白的制备方法，其特征在于，所述方法包含以下步骤：
    ①全基因合成VIP序列；
    ②通过PCR扩增获取HSA序列；
    ③通过限制性内切酶酶切、连接并转化大肠杆菌，获得含编码所述血管活性肠肽的融合蛋白的氨基酸序列的DNA序列的重组表达载体；
    ④将步骤③所述的重组表达载体转化到感受态大肠杆菌TOP10，再转化到宿主表达系统进行表达，即得所述融合蛋白。
  14. 根据权利要求13所述的制备方法，其特征在于，步骤④所述宿主表达系统是酵母。
  15. 根据权利要求14所述的制备方法，其特征在于，所述的酵母为嗜甲醇毕赤酵母。
  16. 一种如权利要求7所述的血管活性肠肽的融合蛋白的制备方法，其特征在于，所述方法包含以下步骤：
    (1)所述血管活性肠肽的融合蛋白的动物表达载体的构建
    ①全基因合成VIP序列；
    ②通过PCR扩增获取HSA序列；
    ③通过限制性内切酶酶切、连接并转化大肠杆菌，获得含编码所述血管活性肠肽的融合蛋白的氨基酸序列的DNA序列的重组动物表达载体；
    (2)所述血管活性肠肽的融合蛋白的酵母表达载体的构建
    ①通过PCR扩增步骤(1)③中重组动物表达载体获取HSA-L2-VIP；
    ②通过限制性内切酶酶切酵母表达载体；
    ③使用融合PCR技术连接(2)①中HSA-L2-VIP与(2)②中酵母表达载体，并转化大肠杆菌，获得含编码所述血管活性肠肽的融合蛋白的氨基酸序列的DNA序列的重组酵母表达 载体；
    (3)将步骤(2)③所述的重组酵母表达载体转化到感受态大肠杆菌TOP10，再转化到酵母中进行表达，即得所述融合蛋白。
  17. 根据权利要求16所述的制备方法，其特征在于，所述的酵母为嗜甲醇毕赤酵母。
  18. 一种含有编码如权利要求1-5和7-9和12中任意一项所述的血管活性肠肽的融合蛋白氨基酸序列的DNA序列的重组表达载体。
  19. 一种含有权利要求18所述的重组表达载体的宿主表达系统。
  20. 权利要求1-5和7-9和12中任意一项所述的血管活性肠肽的融合蛋白在制备抗炎、抗损伤、脑血管疾病、提高睡眠质量的药物中的应用。
  21. 权利要求1-5和7-9和12中任意一项所述的血管活性肠肽的融合蛋白在制备治疗哮喘药物中的应用。
PCT/CN2016/075992 2016-03-09 2016-03-09 一种血管活性肠肽的融合蛋白及其制备方法和应用 WO2017152399A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/075992 WO2017152399A1 (zh) 2016-03-09 2016-03-09 一种血管活性肠肽的融合蛋白及其制备方法和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/075992 WO2017152399A1 (zh) 2016-03-09 2016-03-09 一种血管活性肠肽的融合蛋白及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2017152399A1 true WO2017152399A1 (zh) 2017-09-14

Family

ID=59790081

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/075992 WO2017152399A1 (zh) 2016-03-09 2016-03-09 一种血管活性肠肽的融合蛋白及其制备方法和应用

Country Status (1)

Country Link
WO (1) WO2017152399A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101875923A (zh) * 2009-12-31 2010-11-03 杭州北斗生物技术有限公司 人血清白蛋白与尿酸氧化酶的融合蛋白及其制备方法
CN102596217A (zh) * 2009-08-14 2012-07-18 费斯生物制药公司 经修饰的血管活性肠肽
WO2012170524A1 (en) * 2011-06-06 2012-12-13 Phasebio Pharmaceuticals, Inc. Use of modified vasoactive intestinal peptides in the treatment of hypertension
CN102827290A (zh) * 2012-09-07 2012-12-19 浙江大学 人血清白蛋白与人生长激素脂肪分解结构域的融合蛋白

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596217A (zh) * 2009-08-14 2012-07-18 费斯生物制药公司 经修饰的血管活性肠肽
CN101875923A (zh) * 2009-12-31 2010-11-03 杭州北斗生物技术有限公司 人血清白蛋白与尿酸氧化酶的融合蛋白及其制备方法
WO2012170524A1 (en) * 2011-06-06 2012-12-13 Phasebio Pharmaceuticals, Inc. Use of modified vasoactive intestinal peptides in the treatment of hypertension
CN102827290A (zh) * 2012-09-07 2012-12-19 浙江大学 人血清白蛋白与人生长激素脂肪分解结构域的融合蛋白

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GANEA, D. ET AL.: "The Neuropeptide VIP: Direct Effects on Immune Cells and Involvement in Inflammatory and Autoimmune Diseases", ACTA PHYSIOL., vol. 213, no. 2, 28 February 2015 (2015-02-28), pages 442 - 452, XP055413760 *
SANLIOGLU, A.D. ET AL.: "Therapeutic Potential of VIP vs PACAP in Diabetes", JOURNAL OF MOLECULAR ENDOCRINOLOGY, vol. 49, 18 September 2012 (2012-09-18), pages R157 - R167, XP055413900 *

Similar Documents

Publication Publication Date Title
JP2019073552A (ja) 工学操作された核酸の送達および製剤
CN103998053A (zh) 通过抑制il-4和/或il-13与其各自的受体结合来预防或治疗某些障碍的方法
RU2014137321A (ru) ГИСТИДИЛ-тРНК-СИНТЕТАЗЫ ДЛЯ ЛЕЧЕНИЯ АУТОИММУННЫХ И ВОСПАЛИТЕЛЬНЫХ ЗАБОЛЕВАНИЙ
JP6339231B2 (ja) 線維症性疾患の処置のためのＴｒａｉｌレセプターアゴニスト
CN113307845B (zh) 促进肝细胞增殖和/或抑制肝细胞凋亡的多肽及其用途
CN102863537A (zh) 一种肿瘤靶向性肿瘤坏死因子相关凋亡配体变体及其应用
CN111388476A (zh) 双嘧达莫或其药学上可接受的盐在制备预防和/或治疗肺部炎症药物中的应用
CN107375910A (zh) PTHrP在制备治疗男性性腺功能低下综合征中的应用
CN111423502A (zh) 多肽、其衍生物及其在制备抗肺纤维化的药物中的应用
CN108578709A (zh) 温敏性长效缓释药物载体及其应用
CN106063928B (zh) 一种多肽或其衍生物在治疗高血压性心肌肥厚中的应用
WO2009083880A1 (en) Transglutaminases binding fusion protein, compositions comprising thereof, micro-spheres comprising thereof uses and methods
CN103037887A (zh) 用于治疗呼吸道感染的方法和药物组合物
CN107082804B (zh) 一种卵形鲳鲹β-胸腺素及其应用
CN111777667B (zh) 小肽及其在制备免疫调节药物中的应用
WO2018171535A1 (zh) 重组人胰岛素原融合蛋白及其制备方法和用途
CN107073075A (zh) 用于预防或治疗以异常的成纤维细胞增殖和细胞外基质沉积为特征的疾病、病况或进程的组合物和方法
CN113292656B (zh) 用于防治肥胖的中脑星形胶质细胞源性神经营养因子的融合蛋白
WO2017152399A1 (zh) 一种血管活性肠肽的融合蛋白及其制备方法和应用
CN108929383A (zh) 重组Slit2D2（C386S）-HSA融合蛋白及其在预防和/或治疗肺部炎症中的应用
WO2022247740A1 (zh) 一种多肽及其在制备免疫调节药物中的应用
WO2017152873A1 (zh) 微小rna31前体编码多肽在制备免疫调节药物中的应用
CN106749561B (zh) 一种嗜麦芽窄食单胞菌外膜蛋白及其应用
CN108690123A (zh) 短肽在制备免疫调节药物中的应用
CN110964094B (zh) 人源白细胞蛋白酶抑制因子及其重组制备与应用

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16893050

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 16893050

Country of ref document: EP

Kind code of ref document: A1