WO2024055413A1 - 经修饰的初乳蛋白质组合物及其用途 - Google Patents

经修饰的初乳蛋白质组合物及其用途 Download PDF

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WO2024055413A1
WO2024055413A1 PCT/CN2022/132944 CN2022132944W WO2024055413A1 WO 2024055413 A1 WO2024055413 A1 WO 2024055413A1 CN 2022132944 W CN2022132944 W CN 2022132944W WO 2024055413 A1 WO2024055413 A1 WO 2024055413A1
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colostrum protein
protein composition
modified colostrum
modified
antigen
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俞泽民
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俞泽民
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to a colostrum protein, in particular to a modified colostrum protein composition.
  • Immunoglobulins are proteins mainly secreted by plasma cells and used by the immune system to recognize and neutralize foreign substances, such as pathogens such as bacteria or viruses. Immunoglobulins include IgA, IgD, IgE, IgG and IgM. IgA can be found in breast milk, saliva, tears and mucus in the bronchus. It is very important for mucosal immunity, which is the body's first line of defense against foreign pathogens.
  • Mucosal tissue is the main site of contact and communication between the human body and various antigens in nature, including: 1) oral mucosa composed of multi-layered squamous epithelial cells, nasopharynx (including tonsils) upper end of esophagus, urinary tract and reproductive tract; and 2) single The gastrointestinal mucosa, respiratory mucosa and lower end of the esophagus are composed of layered columnar epithelial cells.
  • the common feature of all mucosal tissues is that they secrete mucus and secretory immunoglobulin A (secretary IgA, SIgA).
  • the organism's response mechanism is to first initiate an intestinal immune response.
  • This immune response mainly stimulates the formation of specific SIgA, and then converts this specificity into SIgA is secreted outside the mucosa, so that the specific SIgA and the antigen form an immune complex (immune complex), thereby preventing the antigen from approaching the mucosa and achieving immune exclusion (immune exclusion).
  • This effect can prevent pathogens from staying in the mucosal layer for a long time, thereby greatly reducing the chance of the pathogen forming a colony.
  • the organism can effectively prevent the formation of bacterial colonies and achieve the function of protecting mucosal tissue through the continuous secretion of specific SIgA.
  • Colostrum also contains immunoglobulins such as IgA, IgD, IgE, IgG and IgM, and these immunoglobulins can play a good defensive role against pathogens such as viruses, bacteria, parasites and yeast.
  • the object of the present invention is to provide a modified colostrum protein composition that has the effect of preventing foreign pathogens.
  • modified colostrum protein composition including: a modified colostrum protein carrier, the amino acid sequence of which is shown in SEQ ID NO: 1; and an antigen.
  • the fragment is connected to the modified colostrum protein carrier.
  • a modified colostrum protein composition is provided, and the amino acid sequence of the antigen fragment is selected from the partial amino acid sequence of pathogenic bacteria and/or viruses that cause mucosal immune diseases.
  • a modified colostrum protein composition is provided, and the amino acid sequence of the antigen fragment is selected from the partial amino acid sequence of a tumor marker.
  • a modified colostrum protein composition is provided, and the modified colostrum protein composition further includes a carrier, excipient or adjuvant.
  • the use of the modified colostrum protein composition as described above is provided for preparing a pharmaceutical composition that induces the mucosal immune system to produce antigen-specific immunoglobulin IgA against the antigen fragment.
  • the organism can promote and enhance the mucosal immune response and improve resistance to the antigen fragment through the modified colostrum protein composition connected with the antigen fragment.
  • the production of antigen-specific immunoglobulin IgA thereby achieving the purpose of preventing infection by specific pathogens.
  • the modified colostrum protein composition of the present invention is used to prepare a pharmaceutical composition that induces the mucosal immune system to produce antigen-specific immunoglobulin IgA against the antigen fragment, so that the modified colostrum protein composition
  • a vaccine-related pharmaceutical composition it provides a more diverse selection of vaccines to prevent diseases caused by various pathogens.
  • Figure 1 shows mice that are not orally fed with yeast containing a modified colostrum protein composition; and are orally fed with the yeast containing the modified colostrum protein composition according to an embodiment of the present invention. Histograms of the absorbance values of immunoglobulin IgA1 and IgA2 in the serum of the mice measured on days 1, 3, 5, and 7 respectively; and
  • Figure 2 shows mice that are orally fed with yeast that does not contain a modified colostrum protein composition; and mice that are orally fed with the yeast that contains the modified colostrum protein composition according to another embodiment of the present invention. Histogram of absorbance values after serial dilution of immunoglobulin IgA in serum of mice after infection on day 3.
  • FIGS. 1 to 2 The embodiment of the present invention will be described below based on FIGS. 1 to 2 . This description is not intended to limit the embodiments of the present invention, but is one example of the embodiments of the present invention.
  • a modified colostrum protein composition according to one embodiment of the present invention includes: a modified colostrum protein carrier, the amino acid sequence of which is shown in SEQ ID NO: 1; and an antigen fragment connected to the modified colostrum protein carrier.
  • the colostrum protein carrier includes: a modified colostrum protein carrier, the amino acid sequence of which is shown in SEQ ID NO: 1; and an antigen fragment connected to the modified colostrum protein carrier.
  • the amino acid sequence of the antigen fragment is selected from the partial amino acid sequence of pathogenic bacteria and/or viruses that cause mucosal immune diseases.
  • the modified colostrum protein composition further includes a carrier, excipient or adjuvant.
  • the modified colostrum protein composition according to an embodiment of the present invention is used to prepare a pharmaceutical composition that induces the mucosal immune system to produce antigen-specific immunoglobulin IgA against the antigen fragment.
  • amino acid sequence of the modified colostrum protein carrier is as shown in SEQ ID NO: 1, which is the 33rd position of the amino acid sequence of the wild-type colostrum protein as shown in SEQ ID NO: 2. It was produced by replacing isoleucine with alanine, glutamic acid at position 101 with cysteine, and arginine at position 175 with cysteine.
  • the modified tertiary structure of the colostrum protein carrier is more stable than that of wild-type colostrum protein.
  • the modified colostrum protein carrier of the present invention is a protein that is purified and bound to peptidoglycan on the cell wall of bacteria, and the protein carrier is obtained by modifying its sequence, and is named Pathogen Identification according to its characteristics. Protein (Pathological Recognition Protein, PRP).
  • the amino acid sequence of the antigen fragment included in the modified colostrum protein composition of the embodiment of the present invention is a partial amino acid sequence of S. suis serotype 2.
  • the amino acid sequence of the antigen fragment included in the modified colostrum protein composition of the present invention is not limited thereto.
  • the amino acid sequence of the antigen fragment can be selected from the amino acid sequences of various pathogens.
  • Streptococcus suis mainly causes meningitis, septicemia, endocarditis, arthritis and even tracheopneumonia in weaned pigs. It can also cause meningitis, endocarditis and septicemia in humans. Other symptoms include arthritis, bronchopneumonia and Fever, which in severe cases can lead to septic shock, disseminated intravascular coagulation, etc., is an important zoonotic disease.
  • Embodiments of the present invention use indirect enzyme-linked immunosorbent assay (Indirect ELISA) to detect the presence of antigen-specific immunoglobulins in serum.
  • the experimental method is as follows:
  • mice of specific pathogen free (SPF) grade were used, and the mice were divided into immune groups (i.e., the mice were orally fed with yeast containing the modified colostrum protein composition).
  • 40 mice each) and a control group i.e., mice that were not orally fed yeast containing the modified colostrum protein composition before immunization
  • each group of 10 mice was divided into 1 group and orally administered Feed the colostrum protein composition inoculated with 25 ⁇ g, and draw blood samples from the mice through the tail vein on the 1st, 3rd, 5th and 7th days after oral feeding of the yeast containing the colostrum protein composition, and use indirect enzyme Combined with immunosorbent analysis, the concentration changes of specific immunoglobulin IgA are detected.
  • 96-well microplates were purified by diluting to 1 ⁇ g/mL with carbonate buffer (15 mmol/1 Na 2 CO 3 , 35 mmol/1 NaHCO 3 [pH 9.6]) at 4°C. Recombinant protein was coated overnight.
  • mice sera of the control group and the immune group were serially diluted using PBST (a two-fold dilution gradient from 1:400 to 1:3276800) and added to the appropriate wells at 100 ⁇ L per well, and the microporous plate was placed on Incubate at 37°C for 1 hour.
  • PBST a two-fold dilution gradient from 1:400 to 1:3276800
  • a secondary antibody peroxidase-conjugated goat anti-mouse IgA antibody (Santa Cruz Biotechnology, CA) was added at a dilution of 1:5000, and 100 ⁇ L per well was added to the appropriate wells. The microwell plate was incubated at 37°C for 30 minutes.
  • microporous plate was incubated with o-phenylenediamine as a substrate for horseradish peroxidase (HRP) to observe the reaction.
  • HRP horseradish peroxidase
  • the end-point titer is defined as the highest dilution of serum at which the absorbance value (OD490) of the serum of the immune group at 490 nm is 2.1 times higher than the average OD490 of the serum of the control group.
  • mice serum from the immune group 100 ⁇ L was added to each well at a dilution of 1:2000.
  • Peroxidase-conjugated goat anti-mouse IgA1 and IgA2 (Santa Cruz Biotechnology) were used as secondary antibodies. Results are expressed as mean ⁇ standard deviation.
  • the specific immunoglobulins IgA1 and IgA2 of the immunized group were higher than those of the control group.
  • the specific immunoglobulins IgA1 and IgA2 were orally fed with yeast containing the modified colostrum protein composition. The highest concentration was reached on the 3rd and 5th days after oral feeding, and dropped rapidly on the 7th day after oral feeding. This may be because the specific immunoglobulin fraction measured on the 1st day after oral feeding was IgM. It is not converted into IgA until the third day, and the rapid decline on the 7th day is because IgA can only exist in the blood for 7 days.
  • the amino acid sequence of the antigen fragment is selected from the partial amino acid sequence of a tumor marker.
  • Another embodiment of the present invention uses an indirect enzyme-linked immunosorbent assay (Indirect ELISA) to detect the presence of antigen-specific immunoglobulins in serum.
  • Indirect ELISA indirect enzyme-linked immunosorbent assay
  • Another embodiment of the present invention divides the entire antigen fragment of Carcinoembryonic Antigen (CEA) into 17 antigen fragments with a length of 9 to 15 amino acids, and selects the most suitable fragments for testing.
  • CEA Carcinoembryonic Antigen
  • the 17 constructed plastids were amplified and cultured in E. coli and then transferred into yeast.
  • the plastid screening conditions were: 1. Plastids that do not produce toxic substances and can grow in yeast; 2. Can grow in URA- Strains grown on culture media; 3. Plastids that have not mutated in sequencing; 4.
  • PRP cancer antigen modified colostrum protein composition
  • mice fed PRP cancer antigen complex peptide The purpose is to detect whether the expression of specific IgA increases after mice are fed PRP cancer antigen complex peptide.
  • C57BL/6J mice were fed empty yeast that did not contain PRP cancer antigen compound peptide and yeast that contained PRP cancer antigen compound peptide.
  • Mice fed empty yeast that did not contain PRP cancer antigen compound peptide belonged to the control group.
  • mice fed yeast containing PRP cancer antigen complex peptide belong to the immune group; the feeding dose is 2500mg/kg, the control group and immune group collected blood from the cheek on the 1st, 3rd and 5th day after feeding Mouse serum was collected. The collected serum samples were tested for IgA neutralizing antibodies by ELISA.
  • the process was as follows: Attach CEA protein to a microporous plate (50ng/well), add diluted serum samples (100 ⁇ l/well) after washing, and wash Then add anti-IgA antibody, wash away excess detection antibody and add HRP coupler. Add HRP substrate to indirectly detect the bound protein, and measure the absorbance value OD 450nm with an ELISA microplate analyzer.
  • Figure 2 shows the results of one mouse in the control group and the immune group on the third day after feeding the respective yeast.
  • the mouse serum was serially diluted to detect the content of IgA neutralizing antibodies.
  • the mice in the immune group were fed yeast containing PRP cancer antigen complex peptide, in the 20-fold dilution of mouse serum, the content of IgA antibodies that specifically recognize CEA antigen fragments was significantly higher.
  • the control group ie, mice fed empty yeast
  • the colostrum protein composition of the present invention can indeed induce mice to produce specific IgA, and the IgA has the ability to recognize CEA antigen.
  • the modified colostrum protein composition in this case can help organisms promote and enhance mucosal immune responses through the antigen fragments connected to the modified colostrum protein carrier, and improve the antigen-specific immune cells against the antigen fragments.
  • the production of protein IgA thereby achieves the purpose of preventing infection by specific pathogens.
  • the modified colostrum protein composition of the present invention is used to prepare a pharmaceutical composition that induces the mucosal immune system to produce antigen-specific immunoglobulin IgA against the antigen fragment, so that the modified colostrum protein composition
  • As a vaccine-related pharmaceutical composition it provides a more diverse selection of vaccines to prevent diseases caused by various pathogens.

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Abstract

本发明涉及一种经修饰的初乳蛋白质组合物，包含：经修饰的初乳蛋白质载体，其氨基酸序列如SEQ ID NO：1所示；以及抗原片段，连接于经修饰的该初乳蛋白质载体。

Description

经修饰的初乳蛋白质组合物及其用途 技术领域
本发明涉及一种初乳蛋白，特别是涉及一种经修饰的初乳蛋白质组合物。
背景技术
免疫球蛋白(抗体)，为一种主要由浆细胞分泌，并可被免疫系统用来识别以及中和外来物质，例如细菌或病毒等病原体的蛋白质。免疫球蛋白包括IgA、IgD、IgE、IgG及IgM，其中IgA可在母乳、唾液、眼泪以及支气管中的黏液被发现，对于作为身体抵御外来病原体的第一道防线的黏膜免疫十分重要。
黏膜组织是人体与自然界各种抗原接触沟通的主要部位，包括：1)多层鳞状上皮细胞构成的口腔黏膜、鼻咽(含扁桃腺)食道上端、泌尿道以及生殖道；与2)单层柱状上皮细胞构成的胃肠黏膜、呼吸道黏膜以及食道下端。所有黏膜组织的共同特征为皆会分泌黏液与分泌性免疫球蛋白A(secretary IgA，SIgA)。
面对肠道细菌及各种颗粒性抗原(particulate antigens)的入侵，生物体的应对机制首先为启动肠道免疫反应，该免疫反应主要是刺激专一性SIgA的形成，再将该专一性SIgA分泌到黏膜外，以使该专一性SIgA与该抗原形成免疫附合体(immune complex)，从而阻止该抗原靠近黏膜，达到免疫阻隔作用(immune exclusion)。此作用能防止病原体在黏膜层长期停留，从而使该病原体形成菌落(colony)的机会随之大幅减少。换言之，生物体通过不断分泌的专一性SIgA可以有效防止菌落形成而达到保护黏膜组织的功能。
雌性哺乳动物于产后2-3天内所初分泌的乳汁称之为初乳(colostrum)，其后再分泌的乳汁则被称为常乳。初乳中亦含有IgA、IgD、IgE、IgG以及IgM等免疫球蛋白，而该些免疫球蛋白对于病毒、细菌、寄生虫及酵母菌等病原体皆能发挥良好的防御作用。
虽然初乳的潜在效益极高，然而，由于初乳的分泌时程过短，以及初乳中的蛋白不稳定、收集不易及保存困难等因素，使得针对初乳蛋白于实际应用层面上的发展至今仍甚为迟缓。
发明内容
因此，本发明的目的即在提供一种经修饰的初乳蛋白质组合物，具有预防外来病原体的功效。
本发明为解决现有技术的问题所采用的技术手段提供一种经修饰的初乳蛋白质组合物，包含：经修饰的初乳蛋白质载体，其氨基酸序列如SEQ ID NO：1所示；以及抗原片段，连接于经修饰的该初乳蛋白质载体。
在本发明的一实施例中提供一种经修饰的初乳蛋白质组合物，该抗原片段的氨基酸序列选自引发黏膜免疫的疾病的致病菌和/或病毒的部分氨基酸序列。
在本发明的一实施例中提供一种经修饰的初乳蛋白质组合物，该抗原片段的氨基酸序列选自肿瘤标记的部分氨基酸序列。
在本发明的一实施例中提供一种经修饰的初乳蛋白质组合物，经修饰的该初乳蛋白质组合物还包括载剂、赋形剂或佐剂。
在本发明的一实施例中提供一种如上述的经修饰的初乳蛋白质组合物用于制备诱导黏膜免疫系统针对该抗原片段而产生抗原专一性免疫球蛋白IgA的医药组成物的用途。
经由本发明的经修饰的初乳蛋白质组合物所采用的技术手段，使得生物体得以通过连接有抗原片段的经修饰的该初乳蛋白质组合物，促进并增强黏膜免疫反应，提高对抗该抗原片段的抗原专一性免疫球蛋白IgA的生成，进而达到预防特定病原体的感染的目的。本发明的经修饰的该初乳蛋白质组合物用于制备诱导黏膜免疫系统针对该抗原片段而产生抗原专一性免疫球蛋白IgA的医药组成物的用途，使经修饰的该初乳蛋白质组合物作为疫苗相关的医药组成物，为预防各式病原体所造成的疾病提供了更多元的疫苗种类选择。
附图说明
图1为显示根据本发明的一实施例的未经口服喂食含有经修饰的初乳蛋白质组合物的酵母菌的小鼠；以及经口服喂食含有经修饰的该初乳蛋白质组合物的该酵母菌后的小鼠于第1、3、5、7天所分别测得的血清中免疫球蛋白IgA1以及IgA2的吸光值直方图；以及
图2为显示根据本发明的另一实施例的经口服喂食未含有经修饰的初乳蛋白质组合物的酵母菌的小鼠；以及经口服喂食含有经修饰的该初乳蛋白质组合物的该酵母菌后的小鼠于第3天所测得的血清中免疫球蛋白IgA，经序列稀释后的吸光值直方图。
具体实施方式
以下根据图1至图2，而说明本发明的实施方式。该说明并非为限制本发明的实施方式，而为本发明的实施例的一种。
依据本发明的一实施例的一种经修饰的初乳蛋白质组合物，包含：经修饰的初乳蛋白质载体，其氨基酸序列如SEQ ID NO：1所示；以及抗原片段，连接于经修饰的该初乳蛋白质载体。
依据本发明的实施例的经修饰的该初乳蛋白质组合物，其中该抗原片段的氨基酸序列选自引发黏膜免疫的疾病的致病菌和/或病毒的部分氨基酸序列。
依据本发明的实施例的经修饰的该初乳蛋白质组合物，其中经修饰的该初乳蛋白质组合物还包括载剂、赋形剂或佐剂。
依据本发明的实施例的经修饰的该初乳蛋白质组合物，用于制备诱导黏膜免疫系统针对该抗原片段而产生抗原专一性免疫球蛋白IgA的医药组成物的用途。
详细而言，经修饰的该初乳蛋白质载体，其氨基酸序列如SEQ ID NO：1所示的氨基酸序列，为自如SEQ ID NO：2所示的野生型初乳蛋白质的氨基酸序列的第33位置的异白氨酸被丙氨酸取代，第101位置的谷氨酸被半胱氨酸取代，以及第175位置的精氨酸被半胱氨酸取代而产生。
进一步而言，经修饰的该初乳蛋白质载体的三级结构相较于野生型初乳蛋白质更为稳定。
详细而言，本发明的经修饰的该初乳蛋白质载体为纯化结合于细菌的细胞壁上的胜肽聚醣的蛋白质，且该蛋白质载体经修饰其序列而得，并依其特性命名为病原辨识蛋白(Pathological Recognition Protein,PRP)。
本发明的实施例的经修饰的该初乳蛋白质组合物所包含的该抗原片段的氨基酸序列为第二型猪链球菌(S.suis serotype 2)的部分氨基酸序列。当然，本发明的经修饰的该初乳蛋白质组合物所包含的该抗原片段的氨基酸序列并不以此为限，该抗原片段的氨基酸序列可选自各式病原体的氨基酸序列。
详细而言，猪链球菌的各血清型中，以第二型最常存于健康带菌或发病猪群，因此较难以针对该菌所造成的疾病进行流行病学分析以及追查感染源。猪链球菌主要引起离乳猪的脑膜炎、败血症、心内膜炎、关节炎甚至之气管肺炎，亦会引起人类的脑膜炎、心内膜炎及败血症，其他症状包括关节炎、支气管肺炎及发热，严重时会导致败血性休克、弥漫性血管内凝血等等，属于重要的人畜共通传染病。
本发明的实施例使用间接酵素结合免疫吸附分析法(Indirect enzyme-linked immunosorbent assay,Indirect ELISA)侦测血清中抗原专一性免疫球蛋白的存在。实验方法如下所示：
采用约4周龄的无特定病原(specific pathogen free,SPF)级雌性BALB/c小鼠，将小鼠分为免疫组(即，经口服喂食含有经修饰的该初乳蛋白质组合物的酵母菌后的小鼠)以及控制组(即，免疫接种前，未经口服喂食含有经修饰的该初乳蛋白质组合物的酵母菌的 小鼠)各40只，每10只小鼠成1组且口服喂食接种25μg的该初乳蛋白质组合物，于口服喂时含有该初乳蛋白质组合物的酵母菌后的第1、3、5以及第7天通过尾静脉抽取小鼠血液样本，并通过间接酵素结合免疫吸附分析法侦测专一性免疫球蛋白IgA的浓度变化。
首先，96孔微量多孔盘(96-well microplates)于4℃下以通过碳酸盐缓冲液(15mmol/1 Na 2CO 3、35mmol/1 NaHCO 3[pH 9.6])稀释至1μg/mL的纯化重组蛋白包覆过夜。
使用含有0.05％Tween-20(PBST)的PBS洗涤3次后，该微量多孔盘于37℃下用含有5％脱脂奶粉的PBST阻断1小时。
随后将使用PBST进行序列稀释(自1:400至1:3276800的两倍稀释梯度)的控制组和免疫组的小鼠血清以每孔100μL添加到适当的孔中，并将该微量多孔盘于37℃下孵育1小时。
洗涤3次后，以1:5000稀释度添加二级抗体(结合过氧化物酶的山羊抗小鼠IgA抗体(Santa Cruz Biotechnology,CA))，以每孔100μL添加到适当的孔中，将该微量多孔盘于37℃下孵育30分钟。
最后，将该微量多孔盘与作为山葵过氧化酶(horseradish peroxidase，HRP)的受质的邻苯二胺一起孵育，以观察反应。
终点效价定义为血清的最高稀释度，此时免疫组的血清于490nm处的吸光值(OD490)比控制组的血清的平均OD 490高2.1倍。
针对IgA1以及IgA2的侦测，以1:2000的稀释度的免疫组的小鼠血清以每孔100μL添加。结合过氧化物酶的山羊抗小鼠IgA1以及IgA2(Santa Cruz Biotechnology)作为二级抗体。结果表示为平均值±标准差。
如图1所示，免疫组的专一性免疫球蛋白IgA1以及IgA2皆高于控制组，专一性免疫球蛋白IgA1以及IgA2于口服喂食含有经修饰的该初乳蛋白质组合物的酵母菌后的第3天以及第5天时达到最高浓度，而口服喂食后的第7天则快速下降，此可能是因为口服喂食后的第1天时所测得的专一性免疫球蛋白部分为IgM，直到第三天方转化为IgA，而第7天时的快速下降是因为IgA只可在血液中存在7天之故。
依据本发明的另一实施例的经修饰的该初乳蛋白质组合物，其中该抗原片段的氨基酸序列选自肿瘤标记的部分氨基酸序列。
本发明的另一实施例使用间接酵素结合免疫吸附分析法(Indirect enzyme-linked immunosorbent assay,Indirect ELISA)侦测血清中抗原专一性免疫球蛋白的存在。实验方法如下所示：
癌胚胎抗原序列片段设计与质体建构：
本发明的另一实施例将癌胚胎抗原(Carcinoembryonic Antigen,CEA)的整个抗原片段分切成17个长度为9～15个氨基酸的抗原片段，从中筛选最适合的片段进行试验。CEA片段经合成后，分别建构在带有经修饰的初乳蛋白质基因的pDONR质体上，所建构的质体最后以定序分析的方式确认序列无误。建构好的17个质体以大肠杆菌放大培养后，再转殖到酵母菌中，质体筛选条件为：1.不产生有毒物质，能在酵母菌生长的质体；2.能在URA-培养基上生长的菌株；3.定序没有发生变异的质体；4.能使小鼠产生较多中和抗体的菌株。最后再以软件分析依据期待电性、结构、稳定度、化学性，找出适合的包含经修饰的初乳蛋白质载体以及CEA抗原片段的经修饰的初乳蛋白质组合物(以下简称为PRP癌抗原复合胜肽)。
IgA中和抗体测试：
目的为检测小鼠喂食PRP癌抗原复合胜肽后，专一性IgA表现量是否增加。C57BL/6J小鼠分别喂食未含有PRP癌抗原复合胜肽的空酵母菌以及含有PRP癌抗原复合胜肽的酵母菌，喂食未含有PRP癌抗原复合胜肽的空酵母菌的小鼠属于控制组，喂食含有PRP癌抗原复合胜肽的酵母菌的小鼠属于免疫组；喂食剂量为2500mg/kg，控制组及免疫组于喂食后第1天、第3天以及第5天以脸颊采血的方式收集小鼠血清。收集的血清检体以ELISA的方式进行IgA中和抗体检测，流程如下：将CEA蛋白附着于微量多孔盘上(50ng/孔)，清洗后加入稀释后的血清检体(100μl/孔)，清洗后加入抗IgA抗体，洗去多余的检测抗体并添加HRP耦合物，添加HRP受质以间接检测结合蛋白，并以ELISA微盘分析仪以吸光值OD 450nm测量。
图2为控制组及免疫组中各一只小鼠于喂食各自的酵母菌后第3天的结果，小鼠血清经序列稀释后检测其IgA中和抗体的含量。详细而言，免疫组的小鼠经喂食含有PRP癌抗原复合胜肽的酵母菌的第3天后，在20倍稀释的小鼠血清中，专一性辨识CEA抗原片段的IgA抗体含量显著的高于控制组(即，喂食空酵母菌的小鼠)，显示本发明的该初乳蛋白质组合物确实能够诱导老鼠产生专一性IgA，且其IgA具有辨识CEA抗原的能力。
本案的经修饰的该初乳蛋白质组合物，能帮助生物体通过经修饰的该初乳蛋白质载体所连接的抗原片段，促进并增强黏膜免疫反应，提高对抗该抗原片段的抗原专一性免疫球蛋白IgA的生成，进而达到预防特定病原体的感染的目的。本发明的经修饰的该初乳蛋白质组合物用于制备诱导黏膜免疫系统针对该抗原片段而产生抗原专一性免疫球蛋白IgA的医药组成物的用途，使经修饰的该初乳蛋白质组合物作为疫苗相关的医药组成物，为预防 各式病原体所造成的疾病提供了更多元的疫苗种类选择。
以上的叙述以及说明仅为本发明的较佳实施例的说明，对于本领域技术人员当可依据以上权利要求书以及上述的说明而作其他的修改，只是这些修改仍应是为本发明的发明精神而在本发明的保护范围中。

Claims (5)

  1. 一种经修饰的初乳蛋白质组合物，其特征在于，包含：
    经修饰的初乳蛋白质载体，其氨基酸序列如SEQ ID NO：1所示；以及
    抗原片段，连接于经修饰的该初乳蛋白质载体。
  2. 根据权利要求1所述的经修饰的初乳蛋白质组合物，其特征在于，所述的抗原片段的氨基酸序列选自引发黏膜免疫的疾病的致病菌和/或病毒的部分氨基酸序列。
  3. 根据权利要求1所述的经修饰的初乳蛋白质组合物，其特征在于，所述的抗原片段的氨基酸序列选自肿瘤标记的部分氨基酸序列。
  4. 根据权利要求1所述的经修饰的初乳蛋白质组合物，其特征在于，经修饰的该初乳蛋白质组合物还包括载剂、赋形剂或佐剂。
  5. 一种将如权利要求1所述的经修饰的初乳蛋白质组合物用于制备诱导黏膜免疫系统针对该抗原片段而产生抗原专一性免疫球蛋白IgA的医药组成物的用途。
PCT/CN2022/132944 2022-09-14 2022-11-18 经修饰的初乳蛋白质组合物及其用途 WO2024055413A1 (zh)

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CN107188946A (zh) * 2016-03-14 2017-09-22 虹广生物科技有限公司 经修饰的初乳蛋白质及其应用
CN108289944A (zh) * 2015-10-07 2018-07-17 勃林格殷格翰动物保健公司 多价猪链球菌多糖-蛋白缀合组合物

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