CN112023038A - 一种复合免疫增强剂 - Google Patents
一种复合免疫增强剂 Download PDFInfo
- Publication number
- CN112023038A CN112023038A CN202010957458.1A CN202010957458A CN112023038A CN 112023038 A CN112023038 A CN 112023038A CN 202010957458 A CN202010957458 A CN 202010957458A CN 112023038 A CN112023038 A CN 112023038A
- Authority
- CN
- China
- Prior art keywords
- immunopotentiator
- bcg
- polysaccharide
- composite
- preparation
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
- 230000000091 immunopotentiator Effects 0.000 title claims abstract description 37
- 150000001875 compounds Chemical class 0.000 title claims description 10
- 150000004676 glycans Chemical class 0.000 claims abstract description 23
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 23
- 239000005017 polysaccharide Substances 0.000 claims abstract description 23
- 239000002131 composite material Substances 0.000 claims abstract description 20
- 108091007433 antigens Proteins 0.000 claims abstract description 16
- 102000036639 antigens Human genes 0.000 claims abstract description 16
- 229960005486 vaccine Drugs 0.000 claims abstract description 12
- 239000000427 antigen Substances 0.000 claims abstract description 11
- 230000002538 fungal effect Effects 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims abstract description 9
- 230000002195 synergetic effect Effects 0.000 claims abstract description 3
- 239000002671 adjuvant Substances 0.000 claims description 19
- 230000036039 immunity Effects 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 241001506047 Tremella Species 0.000 claims description 5
- 230000002708 enhancing effect Effects 0.000 claims description 5
- 241000190633 Cordyceps Species 0.000 claims description 4
- 229920001491 Lentinan Polymers 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 4
- 230000000996 additive effect Effects 0.000 claims description 4
- 229940115286 lentinan Drugs 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 21
- 238000002360 preparation method Methods 0.000 abstract description 14
- 241001465754 Metazoa Species 0.000 abstract description 7
- 230000003308 immunostimulating effect Effects 0.000 abstract description 7
- 230000004936 stimulating effect Effects 0.000 abstract description 6
- 230000000638 stimulation Effects 0.000 abstract description 6
- 230000028993 immune response Effects 0.000 abstract description 5
- 231100000331 toxic Toxicity 0.000 abstract description 5
- 230000002588 toxic effect Effects 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 230000003053 immunization Effects 0.000 abstract description 4
- 238000002649 immunization Methods 0.000 abstract description 4
- 238000013329 compounding Methods 0.000 abstract description 3
- 238000000855 fermentation Methods 0.000 abstract description 3
- 230000004151 fermentation Effects 0.000 abstract description 3
- 239000000568 immunological adjuvant Substances 0.000 abstract description 3
- 241000186366 Mycobacterium bovis Species 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 239000011162 core material Substances 0.000 abstract 1
- 230000000214 effect on organisms Effects 0.000 abstract 1
- 230000008105 immune reaction Effects 0.000 abstract 1
- 230000002163 immunogen Effects 0.000 abstract 1
- 238000007670 refining Methods 0.000 abstract 1
- 230000001235 sensitizing effect Effects 0.000 abstract 1
- 241000233866 Fungi Species 0.000 description 16
- 239000000047 product Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000002158 endotoxin Substances 0.000 description 4
- 229920006008 lipopolysaccharide Polymers 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 241001264174 Cordyceps militaris Species 0.000 description 3
- 101100341519 Homo sapiens ITGAX gene Proteins 0.000 description 3
- 102100022297 Integrin alpha-X Human genes 0.000 description 3
- 240000000599 Lentinula edodes Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 208000025865 Ulcer Diseases 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- 235000001715 Lentinula edodes Nutrition 0.000 description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 2
- 206010040880 Skin irritation Diseases 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 241000908178 Tremella fuciformis Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004362 fungal culture Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 231100000475 skin irritation Toxicity 0.000 description 2
- 230000036556 skin irritation Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000036269 ulceration Effects 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 229920002444 Exopolysaccharide Polymers 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004166 Lanolin Substances 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000001571 immunoadjuvant effect Effects 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229940039717 lanolin Drugs 0.000 description 1
- 235000019388 lanolin Nutrition 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- YGDGZDGRCWHDOU-UHFFFAOYSA-N methyl 4-[[5-chloro-4-(2-hydroxyphenyl)thiophen-2-yl]sulfonylamino]-2-hydroxybenzoate Chemical compound C1=C(O)C(C(=O)OC)=CC=C1NS(=O)(=O)C1=CC(C=2C(=CC=CC=2)O)=C(Cl)S1 YGDGZDGRCWHDOU-UHFFFAOYSA-N 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
一种复合免疫增强剂，以牛结核分枝杆菌(BCG)为基础核心材料，复配具有协同增强免疫作用的辅料——真菌多糖，制备而成的一种免疫佐剂。制备方法为；对发酵培养后的BCG菌体，进行分离、提取、纯化、精制，获得具有可刺激机体非特异性免疫反应的菌体蛋白，再复配加入可溶性真菌多糖，完成复合免疫增强剂的制备。复合免疫增强剂自身为水溶性、具有非特异性免疫刺激功能、对机体组织无毒副作用、无刺激反应、可有效大幅提高免疫接种中，特异性目的抗原的致敏免疫原作用，提高机体对目的抗原的免疫应答反应。该新型复合免疫增强剂可广泛用于动物的抗原免疫、抗体制备、动物疫苗增强，以及人用疫苗的研发使用，具有良好的经济效益和社会效益。
Description
技术领域
本发明属于生物医药制备技术领域。
背景技术
可提高机体免疫增强作用的制剂通常称为“佐剂”，是一种已被长期广泛使用的免疫学试剂，用于试验研究、抗体制备、以及疫苗的作用增强等。其作用原理首先是具有良好的非特异性刺激机体，产生高效率免疫应答反应。辅助特异性致敏原(特异抗原)引发和提高机体对特异抗原的特异性免疫反应。如B细胞反应的特异性抗体的生成，或是T细胞产生的特异性免疫细胞因子。二是能够刺激机体较长时间，对特异性目标抗原保持持续的免疫反应应答。
免疫增强剂(佐剂)已被长期和广泛的应用于免疫学实践中，是公认的传统、经典的免疫学常规试剂。在传统的免疫增强剂(佐剂)中，均是以灭活的BCG菌体为非特异性免疫刺激源，加入乳化的油脂类材料，制备成具有诱发机体非特异性免疫反应，增强对目标抗原的刺激活性，提高特异性抗血清和抗体效价的形成，以及细胞免疫因子的生成。随着试验材料的改进提高，原始使用“羊毛脂+石蜡油乳化”的油脂类载体，被葡聚糖等高分子物质所替代，同样取得了满意的结果和认同。目前知名试剂商Sigma公司的”完全免疫佐剂“就是这类产品。
上述免疫增强剂(佐剂)产品虽显示了在非特异性免疫刺激反应中的作用，但在使用过程中也同时存在一个很难克服的缺陷。因产品中使用了灭活的BCG菌体，具有明显刺激机体的毒副作用。尤其是皮肤、肌肉注射时显现出强烈的红斑、肿块，有些甚至出现溃疡。因此也制约了该类产品的使用范围(特别是在作为疫苗增强作用使用时，因其过强的毒副作用反应，而放弃使用。)。
因此筛选、制备具有同样较强的能够刺激机体非特异性免疫反应，同时又无刺激、无毒副作用，具有良好生物安全性的新型免疫增强剂是一个很好的产品研发思路和课题。
发明内容
本发明目的是解决现有免疫佐剂添加BCG后副作用大的问题，提供一种新的复合免疫增强剂。本发明以精制、纯化的BCG菌体蛋白(BCG菌株做为非特异性免疫刺激的标志菌株，是其它菌属、种菌株很难与其相比和替代的)为新型免疫增强剂的非特异免疫刺激蛋白。加入具有持续、协同增强免疫作用的真菌多糖，制备一种新型的复合免疫增强剂。该新型复合免疫增强剂可广泛用于动物的抗原免疫、抗体制备、动物疫苗增强，以及人用疫苗，可产生良好的经济效益和社会效益。
本发明的技术方案
一种复合免疫增强剂，以分离纯化后的可溶性BCG菌体蛋白为主体加入协同增强免疫作用的辅助材料(分离、纯化的大分子真菌多糖成分)而制成。可溶性BCG菌体蛋白与真菌多糖的复配可选用任何比例进行。
所述BCG菌体蛋白的制作过程是，以发酵培养的牛结核分枝杆菌(BCG)为制备复合免疫增强剂用菌体蛋白的基础菌株，发酵培养的BCG菌体，经超声破碎、超速离心，去除碎片残渣，获得可溶性BCG菌体蛋白，冷冻干燥保存。所获得的可溶性BCG菌体蛋白，经SDS-PAGE检测主要为：100KD、70KD、50KD、35KD和30KD蛋白混合体。
所述真菌多糖为香菇多糖、银耳多糖或虫草多糖等中的一种或多种复配(添加比例可以不同也可以相同)而成，具有免疫刺激作用强，毒副作用小、皮肤刺激反应小的特点。
本发明提供的复合免疫增强剂可作为抗原制备或疫苗的添加剂。如用于动物的抗原免疫、抗体制备、动物疫苗增强，以及人用疫苗等。实际使用过程中，可根据实际需要，在保证生物安全性的前提下，作为疫苗添加剂可使用任一剂量。
本发明的优点和有益效果：
本发明以分离纯化后的BCG菌体蛋白为基础载体，克服了传统佐剂直接添加BCG菌体后副作用大、局部刺激反应大，易引起注射部分红肿、硬结甚至溃烂的缺点。同时，该新型复合免疫增强剂保留了BCG具有的非特异强致敏性，刺激机体免疫的特点，结合大分子真菌多糖，进一步延长了目的抗原蛋白在机体内的留存时间，显著增强了抗原蛋白对机体免疫系统的免疫刺激作用。
附图说明
图1是BCG非特异免疫激活作用；
图2是本发明复合免疫增强剂增强抗体效价，减弱副作用。
具体实施方式
实施例1：BCG蛋白及多糖提取和复合免疫增强剂的制备
1.1材料
1.1.1菌株：所用菌株来源于ATCC提供的牛结核分枝杆菌属(卡介苗，BCG；巴斯德株)(拥有ATCC授权并签有研发、使用协议)。
香菇菌株(Lentinus edodes)、银耳菌株(Tremellafuciformis)、蛹虫草菌株(Cordyceps militaris)由本课题组保存(为商业销售产品)。
1.1.2培养基：购自sigma公司。
其它试剂均采购于天津市中澳天元科技发展有限公司。
1.1.3真菌多糖：香菇多糖、银耳多糖、虫草多糖；本课题组制备。制备方法见1.2.2。
1.2方法
1.2.1菌体(卡介苗，BCG)蛋白提取
牛结核分枝杆菌属(卡介苗，BCG)菌体培养，采用常规标准的液体培养方法获得。经液体培养后的BCG(卡介苗)活菌，菌体浓度为5×107/ml，经分离纯化后，每毫升菌液可获得大约30微克的菌体蛋白。获得的菌体经离心、收集破碎后，超速离心收集上清。冷冻干燥，得菌体蛋白，保存备用。
1.2.2真菌培养及真菌胞外多糖提取
(1)真菌培养基配制：精确称取蛋白胨5g，葡萄糖30g，酵母提取物5g，磷酸二氢钾0.1g，七水硫酸镁0.05g，蒸馏水1000ml，调整pH＝6.5。将液体培养基分装于500ml三角瓶中，115℃高压灭菌20min。将培养基取出，冷却至室温，在无菌条件下加入1ml 1000×维生素母液，1ml 1000×微量元素母液，得到最终的液体真菌培养基，用于后续的真菌菌丝体培养。固体真菌培养基的组成及配制方法与以上方法类似，但在灭菌前加入20g琼脂，灭菌，冷却至40℃后加入维生素及微量元素，分装到试管及培养皿中，得到固体真菌培养基。
(2)真菌培养：将保存于冻干管中的香菇、银耳、蛹虫草三种丝状真菌分别转接到固体真菌培养基。25℃恒温培养箱中培养7-14d后，挑取菌丝，转接到液体真菌培养基，25℃摇床培养7-14d，得到真菌培养液。
(3)真菌胞外多糖提取：将真菌培养液用纱布过滤去除菌丝球，分别得到三种真菌的培养上清。将1L培养上清与3L无水乙醇充分混合均匀，离心分离沉淀。沉淀用无水乙醇洗涤3次，每次乙醇用量50-100mL。将沉淀转移到冷冻真空干燥箱中干燥48h，分别得到香菇多糖、银耳多糖及虫草多糖。
1.2.3将BCG菌体蛋白和真菌多糖按照10:1的比例混匀得到复合免疫增强剂。
实施例2：新型复合免疫增强剂对DC细胞的激活作用
2.1材料：DC 2.4细胞，脂多糖(LPS),CD11C流式抗体
2.1.1将105DC2.4细胞铺于6孔板中，加入2ml的完全培养基。按照实验设计，加入LPS(10ng/ml)作为阳性对照；加入制备的复合免疫增强剂(10μg/ml)作为实验组；加入PBS为对照组。将不同处理组的细胞放于细胞培养箱生长48小时。
2.2收集细胞，并用PBS清洗细胞。加入流式抗体孵育20分钟后上机检测。
2.3结果与结论
CD11C为DC细胞成熟的分子标志物，DC细胞受到刺激成熟后高表达CD11C。DC细胞经新型复合免疫增强剂刺激后CD11C表达明显增强，和LPS阳性对照组表达水平相当(见附图1)。表明，该免疫增强剂对DC细胞有强烈的刺激作用，促进了DC细胞的成熟。
实施例3：新型复合免疫增强剂增强抗体效价，减弱副作用
3.1材料：BalB/c雌鼠，sigma弗氏完全佐剂，抗原蛋白
3.2方法
3.2.1将鼠分为4组，每组3只。分别为新型复合免疫增强剂组，阳性对照组，阴性对照组和空白对照组。在实验环境中喂养一周适应环境后用于实验。
3.2.2配置免疫试剂。新型佐剂组：100μg新型复合免疫增强剂+100μg抗原蛋白，加入PBS至200μl。阳性对照组：100μl sigma佐剂+100μg抗原蛋白，加入PBS至200μl。阴性对照组：100μg抗原蛋白，加入PBS至200μl。空白对照组：PBS 200μl。将配置好的溶剂依次皮内注射于该组小鼠体内。
3.2.3取血，测定免疫效果。4周后尾静脉取血，分离血清，以抗原蛋白包被96孔板，使用ELISA方法测定免疫效果，以OD值表示。同时记录注射点的皮肤变化。无红肿硬结：-，有轻微红肿硬结(<3mm)：+，有较大硬结(≥3mm)或溃烂：++。
3.3结果与结论
新型复合免疫增强剂和sigma传统佐剂具有相似的刺激抗体产生的效果，加入佐剂后抗体的效价水平明显高于仅接种抗原蛋白的对照组。新型复合免疫增强剂组无明显的皮肤刺激症状，sigma传统佐剂组注射部位有明显硬结，副作用较强(见附图2)。表明，新型佐剂可在不引起明显副作用的情况下达到与传统佐剂相当的刺激免疫的效果。
Claims (7)
1.一种复合免疫增强剂，包括可溶性BCG菌体蛋白和协同增强免疫作用的辅助材料。
2.根据权利要求1所述复合免疫增强剂，其特征在于，所述协同增强免疫作用的辅助材料是大分子真菌多糖。
3.根据权利要求2所述复合免疫增强剂，其特征在于，所述大分子真菌多糖为香菇多糖、银耳多糖或虫草多糖中的一种或多种。
4.根据权利要求3所述复合免疫增强剂，其特征在于，多种真菌多糖同时添加使用时，添加比例可以不同也可以相同。
5.根据权利要求1至4任一项所述复合免疫增强剂，其特征在于，可溶性BCG菌体蛋白与协同增强免疫作用的辅料材料大分子真菌多糖的复配可选用任何比例进行。
6.权利要求1至4任一项所述复合免疫增强剂的应用，作为抗原制备或疫苗的添加剂。
7.根据权利要求6所述的应用，其特征在于，实际使用过程中，可根据实际需要，在保证生物安全性的前提下，作为疫苗添加剂可使用任一剂量。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010957458.1A CN112023038B (zh) | 2020-09-13 | 2020-09-13 | 一种复合免疫增强剂 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010957458.1A CN112023038B (zh) | 2020-09-13 | 2020-09-13 | 一种复合免疫增强剂 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112023038A true CN112023038A (zh) | 2020-12-04 |
| CN112023038B CN112023038B (zh) | 2023-05-26 |
Family
ID=73589034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010957458.1A Active CN112023038B (zh) | 2020-09-13 | 2020-09-13 | 一种复合免疫增强剂 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112023038B (zh) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4439199A (en) * | 1980-02-19 | 1984-03-27 | Alza Corporation | Method for administering immunopotentiator |
| US5116612A (en) * | 1987-06-23 | 1992-05-26 | Allergy Immuno Technologies, Inc. | Immunotherapy agents for treatment of IgE mediated allergies |
| CN103463634A (zh) * | 2013-09-09 | 2013-12-25 | 镇江万山红遍农业园 | 免疫增强剂及其使用方法 |
| CN103550244A (zh) * | 2013-11-11 | 2014-02-05 | 辽宁医学院 | 一种具有协同增强体液和细胞免疫作用的中药复合多糖及应用 |
| US20160144008A1 (en) * | 2001-09-03 | 2016-05-26 | Glycanova As | Production of fungal extracellular immune stimulating compounds |
| WO2018047797A1 (ja) * | 2016-09-06 | 2018-03-15 | セルメディシン株式会社 | 免疫刺激剤 |
| CN109666609A (zh) * | 2019-01-14 | 2019-04-23 | 付家栋 | 一种赤红球菌发酵方法及其作为佐剂在动物疫苗中的应用 |
-
2020
- 2020-09-13 CN CN202010957458.1A patent/CN112023038B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4439199A (en) * | 1980-02-19 | 1984-03-27 | Alza Corporation | Method for administering immunopotentiator |
| US5116612A (en) * | 1987-06-23 | 1992-05-26 | Allergy Immuno Technologies, Inc. | Immunotherapy agents for treatment of IgE mediated allergies |
| US20160144008A1 (en) * | 2001-09-03 | 2016-05-26 | Glycanova As | Production of fungal extracellular immune stimulating compounds |
| CN103463634A (zh) * | 2013-09-09 | 2013-12-25 | 镇江万山红遍农业园 | 免疫增强剂及其使用方法 |
| CN103550244A (zh) * | 2013-11-11 | 2014-02-05 | 辽宁医学院 | 一种具有协同增强体液和细胞免疫作用的中药复合多糖及应用 |
| WO2018047797A1 (ja) * | 2016-09-06 | 2018-03-15 | セルメディシン株式会社 | 免疫刺激剤 |
| CN109666609A (zh) * | 2019-01-14 | 2019-04-23 | 付家栋 | 一种赤红球菌发酵方法及其作为佐剂在动物疫苗中的应用 |
Non-Patent Citations (4)
| Title |
|---|
| DRANDARSKA等: ""Combined immunomodulating effects of BCG and Lentinan after intranasal application in guinea pigs"", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
| 刘朝阳等: "生物反应调节剂在抗肿瘤作用中的应用", 《医学研究杂志》 * |
| 常景玲等: "《天然生物活性物质及其制备技术》", 31 August 2007 * |
| 韩瑞发等: "《实践探索》", 31 May 2008 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112023038B (zh) | 2023-05-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lancefield et al. | Type-specific polysaccharide antigens of group B streptococci | |
| US9249438B2 (en) | Production of fungal extracellular immune stimulating compounds | |
| KR102195870B1 (ko) | 차가버섯균 및 그의 응용 | |
| CN101830968B (zh) | 一种具有免疫调节作用的法氏囊七肽 | |
| JP2003081863A (ja) | 楠茸の菌糸体生物活性物質、その組成物質及びその製造方法 | |
| Mizuno | Development of an antitumor biological response modifier from Phellinus linteus (Berk. et Curt.) Teng (Aphyllophoromycetideae) | |
| CN101805763B (zh) | 一种具有增强免疫作用的桦褐孔菌胞内外混合粗多糖 | |
| Bisko et al. | Effects of cultivation parameters on intracellular polysaccharide production in submerged culture of the edible medicinal mushroom Lentinula edodes. | |
| JPH0561255B2 (zh) | ||
| CN110484459B (zh) | 一株植物乳杆菌及其应用 | |
| US4663438A (en) | Novel nucleic acid-containing glycoprotein | |
| CN107550946B (zh) | 一种家禽免疫增强剂的制备及应用 | |
| CN112023038B (zh) | 一种复合免疫增强剂 | |
| KR0174433B1 (ko) | 펠리누스 린테우스로부터 분리된 항암 면역활성 다당류 및 이의 제조 방법 | |
| CN110591986B (zh) | 一株可缓解类风湿性关节炎的干酪乳杆菌及其应用 | |
| AU2020103625A4 (en) | Applications of bacillus calmette-guerin (bcg) polysaccharide and nucleic acid in in-vitro culture of cytokine-induced-killer (cik) cells and preparation of antitumor drugs | |
| KR20020079238A (ko) | 항비듬균 면역항체와 그 용도 | |
| CN107007831A (zh) | 乙型肝炎dna疫苗的免疫佐剂 | |
| US4288557A (en) | Antigenic complex from N. gonorrhoeae | |
| CN109705194B (zh) | 一种促进aiv和/或ndv疫苗的免疫反应的法氏囊活性五肽及应用 | |
| GB2077101A (en) | Antiallergic composition containing streptococci | |
| CN110835378A (zh) | 灵芝及酵母β-葡聚糖复合多糖体组合物 | |
| RU2023445C1 (ru) | Способ получения иммуномодулятора | |
| JP4280431B2 (ja) | 抗ニキビ用組成物及びそれを含有するニキビ用化粧料 | |
| TSUMITA et al. | The nature of the Middlebrook-Dubos haemagglutination antigen of Mycobacterium tuberculosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220727 Address after: 300000 No. 23, Pingjiang Road, Hexi District, Tianjin Applicant after: TIANJIN INSTITUTE OF UROLOGY Applicant after: Panru Biotechnology (Tianjin) Co.,Ltd. Address before: 300000 No. 23, Pingjiang Road, Hexi District, Tianjin Applicant before: TIANJIN INSTITUTE OF UROLOGY |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240531 Address after: Building 1-301-A324, Building 8, Huiying Industrial Park, No. 86 Zhonghuan West Road, Tianjin Binhai New Area Pilot Free Trade Zone (Airport Economic Zone), 300000 RMB Patentee after: Panru Biotechnology (Tianjin) Co.,Ltd. Country or region after: China Address before: 300000 No. 23, Pingjiang Road, Hexi District, Tianjin Patentee before: TIANJIN INSTITUTE OF UROLOGY Country or region before: China Patentee before: Panru Biotechnology (Tianjin) Co.,Ltd. |