复方蜂胶纳米乳佐剂的研制
摘要
本研究将蜂胶和黄芪多糖复配,以纳米乳为载体,制备出一种复方蜂胶纳米乳佐剂,目的在于改良常规油佐剂存在的问题。通过对该新型佐剂稳定性、安全性和免疫效力考察,为该佐剂在预防兽医中的应用提供理论依据。
第一部分:复方蜂胶纳米乳佐剂的制备及其质量评价。利用伪三元相图法筛选处方,以RH-40、无水乙醇、乙酸乙酯、蜂胶和黄芪多糖为原料制备了复方蜂胶纳米乳,并对其结构类型、形态粒径、黏度、稳定性进行考察。结果表明:复方蜂胶纳米乳透射电镜下呈球形,平均粒径为12.70 nm,PDI为0.188,粒径分布均匀;黏度为2s·0.4 mL-1;该制剂体系稳定。复方蜂胶纳米乳佐剂制备工艺简单、稳定性好、黏度低,符合生物制品质量检验的要求。
第二部分:复方蜂胶纳米乳佐剂的安全性评价。本研究采用急性经口毒性试验、急性眼刺激试验、肌肉刺激性试验、热原试验和细胞毒性试验考察了复方蜂胶纳米乳佐剂的安全性。结果表明:复方蜂胶纳米乳佐剂LD50>5000 mg/kg,属实际无毒;复方蜂胶纳米乳佐剂对眼和肌肉均无刺激性;热原试验表明,复方蜂胶纳米乳佐剂符合规定;复方蜂胶纳米乳佐剂稀释100倍以上无细胞毒性。复方蜂胶纳米乳佐剂安全性高,对活体无刺激,符合生物制品安全检验的要求。
第三部分:复方蜂胶纳米乳佐剂灭活苗的制备及攻毒保护试验。本研究制备出复方蜂胶纳米乳佐剂-H9亚型禽流感病毒灭活苗,并对该疫苗进行了质量和安全性评价,同时进行了攻毒保护试验。结果表明,该灭活疫苗的制备采用直接配苗法,简单易操作;质量评价结果显示,蜂胶外观澄清透明,结构类型为O/W型;高速离心试验5000r/min,离心30min,无破乳现象发生;黏度测定2.5s·0.4 mL-1;无菌检验符合规定;安全检验结果显示,抗原灭活彻底,无血凝活性,疫苗接种鸡只无不良反应;攻毒保护率100%。新型禽流感灭活疫苗物理性状稳定、黏度适宜注射、安全可靠,为该佐剂在生产实践中的应用提供可靠依据。
第四部分:复方蜂胶纳米乳佐剂的免疫效力研究。本章在前期工作基础上,通过HA-HI和MTT淋巴细胞转化试验考察复方蜂胶纳米乳佐剂-H9亚型禽流感灭活苗的体液免疫水平和细胞免疫水平。结果显示:当佐剂中蜂胶浓度达到15mg/mL时,复方蜂胶纳米乳佐剂疫苗免疫效果显著,能够诱导较高的抗体水平,并能有效激活机体的细胞免疫应答;与白油疫苗组相比,该新型疫苗免疫后产生抗体时间更早、滴度更高;淋转试验结果显示,复方蜂胶纳米乳佐剂灭活苗细胞免疫产生迅速,细胞免疫水平高于白油疫苗组、抗原对照组。由此可见,复方蜂胶纳米乳佐剂具有广阔的开发前景。
In order to modify the disadvantages of oil adjuvant, we prepared a new kind of compound propolis nanoemulsion adjuvant. Through stability experiment, security experiment, as well as immune effectiveness experiment, we provided the theoretical basis for the application of compound propolis nanoemulsion adjuvant in preventive veterinary medicine.
Part I: The preparation and quality evaluation of Compound propolis nanoemulsion adjuvant. This new form adjuvant was prepared with the method of pseudoternary phase diagrams. RH-40, absolute alcohol, ethyl acetate, propolis, astragalus polysaccharides and distilled water were selected to prepare compound propolis nanoemulsion. Its structural type, appearance, particle size, viscosity, and physical stability were tested respectively. TEM and Nicomp388/Zeta PALS presented the compound propolis nanoemulsion as spherical drops with uniform size distribution and mean diameter of 12.70 nm. PDI is 0.188.The viscosity of compound propolis nanoemulsion was 2s·0.4 mL-1, and good physical stability was proved by stability test. Compound propolis nanoemulsion adjuvant met the requirements of quality of biological products on account of its simple preparation, high stability, low viscosity.
Part II: The security evaluation of compound propolis nanoemulsion adjuvant. In this section,we evaluated the safety of compound propolis nanoemulsion adjuvant, through acute toxicity test, eye irritation test, muscle stimulation test, pyrogen test and cytotoxicity test.. The results showed that compound propolis nanoemulsion adjuvant was truly nontoxic, LD50>5000. Compound propolis nanoemulsion adjuvant had no eye irritation and muscle stimulation test, pyrogen inspection conforms to the regulation. Cytotoxicity test showed that there was no significant toxicity to L929 in this adjuvant diluted more than 100 times. Compound propolis nanoemulsion adjuvant met the safety requirements of biological products on account of its high security, low irritation.
Part III: The preparation and protective effect of compound propolis nanoemulsion adjuvant inactivated vaccine. In this section, we prepared AIV-H9N2 compound propolis nanoemulsion adjuvant inactivated vaccine, whose quality and security was evaluated. The results showed that the preparation of this kind of vaccine was easy to operate. The prepared vaccine was the type of oil-in-water, transparent and homogeneous, without emulsion breaking phenomenon by centrifuge test at 5000r/min for 30min. The viscosity of vaccine was 2.5s·0.4 mL-1, and the sterility test result met quality standards. The security tests showed that the AIV antigen was completely inactivated, without hemagglutination activity. The vaccine had no adverse reactions to immunized chickens, and protection rate was 100%. This new kind of AIV vaccine was stable, safe and viscosity suitable for injection. So it met requirements of biological products.
Part IV: The immune efficacy test of compound propolis nanoemulsion adjuvant. Based on the preliminary studies, humoral immunity and cellular immunity were tested respectively by HI and lymphocyte transformation. The results as follows: When propolis reached the concentration of 15mg/mL, compound propolis nanoemulsion adjuvant inactivated vaccine had a significant immune effect. Compound propolis nanoemulsion adjuvant inactivated vaccine group produced antibody earlier and higher than that of oil adjuvant vaccine group;Compared with white oil adjuvant vaccine group and blank control group, compound propolis nanoemulsion adjuvant inactivated vaccine could stimulate the body to generate stronger cellular immunity. The results implied this new kind of adjuvant had a broad development prospects.
第一部分:复方蜂胶纳米乳佐剂的制备及其质量评价。利用伪三元相图法筛选处方,以RH-40、无水乙醇、乙酸乙酯、蜂胶和黄芪多糖为原料制备了复方蜂胶纳米乳,并对其结构类型、形态粒径、黏度、稳定性进行考察。结果表明:复方蜂胶纳米乳透射电镜下呈球形,平均粒径为12.70 nm,PDI为0.188,粒径分布均匀;黏度为2s·0.4 mL-1;该制剂体系稳定。复方蜂胶纳米乳佐剂制备工艺简单、稳定性好、黏度低,符合生物制品质量检验的要求。
第二部分:复方蜂胶纳米乳佐剂的安全性评价。本研究采用急性经口毒性试验、急性眼刺激试验、肌肉刺激性试验、热原试验和细胞毒性试验考察了复方蜂胶纳米乳佐剂的安全性。结果表明:复方蜂胶纳米乳佐剂LD50>5000 mg/kg,属实际无毒;复方蜂胶纳米乳佐剂对眼和肌肉均无刺激性;热原试验表明,复方蜂胶纳米乳佐剂符合规定;复方蜂胶纳米乳佐剂稀释100倍以上无细胞毒性。复方蜂胶纳米乳佐剂安全性高,对活体无刺激,符合生物制品安全检验的要求。
第三部分:复方蜂胶纳米乳佐剂灭活苗的制备及攻毒保护试验。本研究制备出复方蜂胶纳米乳佐剂-H9亚型禽流感病毒灭活苗,并对该疫苗进行了质量和安全性评价,同时进行了攻毒保护试验。结果表明,该灭活疫苗的制备采用直接配苗法,简单易操作;质量评价结果显示,蜂胶外观澄清透明,结构类型为O/W型;高速离心试验5000r/min,离心30min,无破乳现象发生;黏度测定2.5s·0.4 mL-1;无菌检验符合规定;安全检验结果显示,抗原灭活彻底,无血凝活性,疫苗接种鸡只无不良反应;攻毒保护率100%。新型禽流感灭活疫苗物理性状稳定、黏度适宜注射、安全可靠,为该佐剂在生产实践中的应用提供可靠依据。
第四部分:复方蜂胶纳米乳佐剂的免疫效力研究。本章在前期工作基础上,通过HA-HI和MTT淋巴细胞转化试验考察复方蜂胶纳米乳佐剂-H9亚型禽流感灭活苗的体液免疫水平和细胞免疫水平。结果显示:当佐剂中蜂胶浓度达到15mg/mL时,复方蜂胶纳米乳佐剂疫苗免疫效果显著,能够诱导较高的抗体水平,并能有效激活机体的细胞免疫应答;与白油疫苗组相比,该新型疫苗免疫后产生抗体时间更早、滴度更高;淋转试验结果显示,复方蜂胶纳米乳佐剂灭活苗细胞免疫产生迅速,细胞免疫水平高于白油疫苗组、抗原对照组。由此可见,复方蜂胶纳米乳佐剂具有广阔的开发前景。
In order to modify the disadvantages of oil adjuvant, we prepared a new kind of compound propolis nanoemulsion adjuvant. Through stability experiment, security experiment, as well as immune effectiveness experiment, we provided the theoretical basis for the application of compound propolis nanoemulsion adjuvant in preventive veterinary medicine.
Part I: The preparation and quality evaluation of Compound propolis nanoemulsion adjuvant. This new form adjuvant was prepared with the method of pseudoternary phase diagrams. RH-40, absolute alcohol, ethyl acetate, propolis, astragalus polysaccharides and distilled water were selected to prepare compound propolis nanoemulsion. Its structural type, appearance, particle size, viscosity, and physical stability were tested respectively. TEM and Nicomp388/Zeta PALS presented the compound propolis nanoemulsion as spherical drops with uniform size distribution and mean diameter of 12.70 nm. PDI is 0.188.The viscosity of compound propolis nanoemulsion was 2s·0.4 mL-1, and good physical stability was proved by stability test. Compound propolis nanoemulsion adjuvant met the requirements of quality of biological products on account of its simple preparation, high stability, low viscosity.
Part II: The security evaluation of compound propolis nanoemulsion adjuvant. In this section,we evaluated the safety of compound propolis nanoemulsion adjuvant, through acute toxicity test, eye irritation test, muscle stimulation test, pyrogen test and cytotoxicity test.. The results showed that compound propolis nanoemulsion adjuvant was truly nontoxic, LD50>5000. Compound propolis nanoemulsion adjuvant had no eye irritation and muscle stimulation test, pyrogen inspection conforms to the regulation. Cytotoxicity test showed that there was no significant toxicity to L929 in this adjuvant diluted more than 100 times. Compound propolis nanoemulsion adjuvant met the safety requirements of biological products on account of its high security, low irritation.
Part III: The preparation and protective effect of compound propolis nanoemulsion adjuvant inactivated vaccine. In this section, we prepared AIV-H9N2 compound propolis nanoemulsion adjuvant inactivated vaccine, whose quality and security was evaluated. The results showed that the preparation of this kind of vaccine was easy to operate. The prepared vaccine was the type of oil-in-water, transparent and homogeneous, without emulsion breaking phenomenon by centrifuge test at 5000r/min for 30min. The viscosity of vaccine was 2.5s·0.4 mL-1, and the sterility test result met quality standards. The security tests showed that the AIV antigen was completely inactivated, without hemagglutination activity. The vaccine had no adverse reactions to immunized chickens, and protection rate was 100%. This new kind of AIV vaccine was stable, safe and viscosity suitable for injection. So it met requirements of biological products.
Part IV: The immune efficacy test of compound propolis nanoemulsion adjuvant. Based on the preliminary studies, humoral immunity and cellular immunity were tested respectively by HI and lymphocyte transformation. The results as follows: When propolis reached the concentration of 15mg/mL, compound propolis nanoemulsion adjuvant inactivated vaccine had a significant immune effect. Compound propolis nanoemulsion adjuvant inactivated vaccine group produced antibody earlier and higher than that of oil adjuvant vaccine group;Compared with white oil adjuvant vaccine group and blank control group, compound propolis nanoemulsion adjuvant inactivated vaccine could stimulate the body to generate stronger cellular immunity. The results implied this new kind of adjuvant had a broad development prospects.
引文
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