SPF鸡胚与低免鸡胚在禽流感灭活疫苗生产中的应用研究
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摘要
之前有很多报道表明新城疫病毒在SPF鸡胚中增殖病毒的效果要远远高于在普通低免鸡胚,对于禽流感病毒在SPF鸡胚和低免鸡胚之间的对比还比较少见。而本试验以重组禽流感病毒(H5N1亚型Re-5株)为研究对象,模拟禽流感灭活疫苗生产,通过以SPF鸡胚和普通生产用低免鸡胚分别为生产原料制备灭活油乳苗,从生产的各个阶段到最后成品的免疫效果进行全程进行数据对比分析。从SPF鸡胚与普通生产用低免鸡胚母源抗体及尿囊液无菌检验对比可知普通生产用低免鸡胚存在不同程度的母源抗体而SPF鸡胚均不存在,普通生产用低免鸡胚三个抽检结果中仅1/3无菌生长,而SPF鸡胚的三个批次抽检结果均为3/3无菌生长。通过不同种毒稀释倍数接种SPF鸡胚试验可知,接种SPF鸡胚所用种毒的稀释倍数选取100万为最佳,可以达到灭活疫苗生产用毒株的标准,并且可提高种蛋的利用率,降低生产成本。符合禽流感病毒低致病力特征,对禽类和哺乳动物高度安全,免疫效力高,且适合利用鸡胚大量增殖。本试验采用全手工接种、收获的鸡胚尿囊液,经过初滤去除大分子的杂质,以避免尿囊液中大颗粒分子对灭活剂的消耗。为了能尽可能减少灭活剂甲醛的添加量而又能保证抗原的灭活检验及无菌检验合格,对常规抗原和试验抗原抽样,分别选取0.15%和0.20%两个甲醛浓度进行灭活,以SPF鸡胚生产的试验抗原能够比以普通生产用低免鸡胚生产的常规抗原以更低的甲醛含量达到灭活的目的。半成品检验数据对比中试验抗原灭活前HA、灭活后HA及EID50三项数据均优于常规抗原。以普通生产用低免鸡胚和SPF鸡胚为生产原料模拟禽流感灭活疫苗操作工艺生产抗原并加以各种乳化剂,按照配方的设计,用乳化泵进行充分搅拌制成的油乳剂灭活疫苗,经过物理性状检验,所制的两种疫苗均是乳白色的油包水剂型,粘度小、均一稳定。通过无菌和安全检验,鸡只均无发生任何不良影响,证明两种疫苗对检验鸡均安全。以SPF鸡胚生产的试验灭活疫苗与以普通生产用低免鸡胚生产的常规灭活疫苗相比,各部分HI抗体水平均高于后者。经免疫持续期抗体消长规律的分析发现,试验疫苗HI抗体产生更为稳定,免疫应答符合油乳剂灭活疫苗的要求。通过原料检验、半成品检验和成品检验三个阶段进行数据对比,为目前我国禽流感灭活疫苗生产中提高抗原效价提供技术参考,为今后禽流感灭活疫苗的发展、提高质量寻求新的途径提供试验数据,促进我国兽用生物制品的研制和开发。
There are many reports that before the Newcastle disease virus in SPF chicken embryos results in the proliferation of the virus is much higher than those in ordinary low-free chicken embryo, for avian influenza virus in SPF chicken-free chicken and low contrast between the still relatively rare. And this experiment with recombinant avian influenza virus (H5N1 subtype Re-5 strain) as the research object to simulate avian influenza inactivated vaccine production, through SPF chicken embryos were used and the general production of raw materials for the production of inactivated oil emulsion preparation of seedlings , from production to the final product at all stages of the immune effect of a full course of the data were analyzed. SPF chicken embryos with the normal production of maternal antibodies and allantoic fluid sterile test comparison we can see normal production chicken varying degrees of maternal antibodies do not exist in SPF chicken embryos, normal embryo production results of three sampling only 1 / 3 sterile growth, while the SPF chicken sampling results of the three batches were 3 / 3 sterile growth. Through different kinds of drug dilutions inoculated SPF chicken embryo test we can see that SPF chicken embryos inoculated with the kinds of poisoning by selected dilution of 1 / 1 million as the best inactivated vaccine production can be achieved with the standard strains, and can improve the use of egg rate, lower production costs. Consistent with a low pathogenicity avian influenza virus features, pairs of birds and mammals, a high degree of safety, immunization of high effectiveness, and suitable for the use of a large number of the proliferation of chick embryo. This test uses hand-inoculation, harvested chorioallantoic fluid, filtered through the first impurity addition to macromolecules in order to avoid large particles allantoic fluid molecules inactivated agent consumption. In order to minimize the formaldehyde inactivated agent to add volume but also ensures that the inactivation of antigen test and sterile test pass tests of conventional antigen and sample antigen, respectively, 0.15% and 0.20% select two concentrations of formaldehyde inactivated, in SPF chicken embryos can be produced by testing antigen production than the ordinary chicken embryo production of conventional antigen to achieve a lower formaldehyde content inactivated purposes. Semi-pilot test data comparison before antigen inactivate HA, inactivated HA and EID50 3 data are superior to conventional antigen. Ordinary production and SPF chicken embryos for the production of raw materials simulated operation of avian influenza inactivated vaccine antigen and make a variety of craft production emulsifier, in accordance with the design formula, mixing with the emulsion pump sufficient oil emulsion made from inactivated vaccine, After physical properties testing, the system of two kinds of vaccines are milky white water in oil formulations, the viscosity is small, uniform stability. Through the sterile and safety inspection, no chickens any adverse effects to prove that the two vaccines are safe for testing the chicken. SPF chicken embryos to produce the pilot production of inactivated vaccine with an ordinary chick embryo production of conventional inactivated vaccine compared to the various parts of HI antibody were higher than the latter. The duration of the immune antibody Variation of the analysis found that the test vaccine is more stable HI antibody immune response in line with the requirements of oil-emulsion inactivated vaccine. Through the inspection of raw materials, semi-finished and finished product testing in three stages of testing data comparison for the present, China's Production of avian influenza inactivated vaccine antigen titer increased provision of technical reference for the future development of avian influenza inactivated vaccine, to seek new ways to improve the quality to provide test data, promoting China's veterinary biological products research and development.
There are many reports that before the Newcastle disease virus in SPF chicken embryos results in the proliferation of the virus is much higher than those in ordinary low-free chicken embryo, for avian influenza virus in SPF chicken-free chicken and low contrast between the still relatively rare. And this experiment with recombinant avian influenza virus (H5N1 subtype Re-5 strain) as the research object to simulate avian influenza inactivated vaccine production, through SPF chicken embryos were used and the general production of raw materials for the production of inactivated oil emulsion preparation of seedlings , from production to the final product at all stages of the immune effect of a full course of the data were analyzed. SPF chicken embryos with the normal production of maternal antibodies and allantoic fluid sterile test comparison we can see normal production chicken varying degrees of maternal antibodies do not exist in SPF chicken embryos, normal embryo production results of three sampling only 1 / 3 sterile growth, while the SPF chicken sampling results of the three batches were 3 / 3 sterile growth. Through different kinds of drug dilutions inoculated SPF chicken embryo test we can see that SPF chicken embryos inoculated with the kinds of poisoning by selected dilution of 1 / 1 million as the best inactivated vaccine production can be achieved with the standard strains, and can improve the use of egg rate, lower production costs. Consistent with a low pathogenicity avian influenza virus features, pairs of birds and mammals, a high degree of safety, immunization of high effectiveness, and suitable for the use of a large number of the proliferation of chick embryo. This test uses hand-inoculation, harvested chorioallantoic fluid, filtered through the first impurity addition to macromolecules in order to avoid large particles allantoic fluid molecules inactivated agent consumption. In order to minimize the formaldehyde inactivated agent to add volume but also ensures that the inactivation of antigen test and sterile test pass tests of conventional antigen and sample antigen, respectively, 0.15% and 0.20% select two concentrations of formaldehyde inactivated, in SPF chicken embryos can be produced by testing antigen production than the ordinary chicken embryo production of conventional antigen to achieve a lower formaldehyde content inactivated purposes. Semi-pilot test data comparison before antigen inactivate HA, inactivated HA and EID50 3 data are superior to conventional antigen. Ordinary production and SPF chicken embryos for the production of raw materials simulated operation of avian influenza inactivated vaccine antigen and make a variety of craft production emulsifier, in accordance with the design formula, mixing with the emulsion pump sufficient oil emulsion made from inactivated vaccine, After physical properties testing, the system of two kinds of vaccines are milky white water in oil formulations, the viscosity is small, uniform stability. Through the sterile and safety inspection, no chickens any adverse effects to prove that the two vaccines are safe for testing the chicken. SPF chicken embryos to produce the pilot production of inactivated vaccine with an ordinary chick embryo production of conventional inactivated vaccine compared to the various parts of HI antibody were higher than the latter. The duration of the immune antibody Variation of the analysis found that the test vaccine is more stable HI antibody immune response in line with the requirements of oil-emulsion inactivated vaccine. Through the inspection of raw materials, semi-finished and finished product testing in three stages of testing data comparison for the present, China's Production of avian influenza inactivated vaccine antigen titer increased provision of technical reference for the future development of avian influenza inactivated vaccine, to seek new ways to improve the quality to provide test data, promoting China's veterinary biological products research and development.
引文
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[9]章金钢.病毒活载体疫苗的研究进展[J].中国兽药杂志, 1994, 28(2): 37-39.
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[12]王锡,唐秀英,李金璋,等.禽流感诊断技术[M].中国农业科学院哈尔滨兽医研究所, 1994, 10: 84-85.
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[17]阴天榜,刘兴友.家禽免疫学[M].北京:中国农业出版社, 1999, 10: 202-209.
[18]卡尔尼克, B W.禽病学(第十版)[M].北京:中国农业出版社, 1999: 691-712.
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[21]孟良玉,王志亮,魏荣,等.我国新城疫病毒分离株分子生物学特性和基因型的研究[M].中国畜牧兽医学会禽病学分会第十一次研讨论文集. 2002, 10: 35-36.
[22]蔡宝祥主编,家禽传染病(第二版)[M].北京:中国农业出版社, 2001: 304-301.
[23]金扩世,金宁一,王兴龙,等.新城疫病毒弱毒疫苗的免疫研究[J].动物科学与动物医学, 2000, 17(4): 31-32.
[24]郝勤宗,李占雷,侯艳红,等.新城疫热稳定性天然弱毒株B95免疫效果的研究[J].河北农业大学学报, 2001, 24(1): 53-55.
[25]丁壮,殷震.新城疫病毒FIN蛋白基因的g隆、序列分析、表达及其重组蛋白免疫原性研究[D].解放军军需大学博士论文, 2002: 87-92.
[26]潘志明,焦新安,张小荣,等.运送鹅源新城疫病毒DNA疫苗的减毒沙门氏菌及其免疫原性[J].农业生物技术学报, 2004, 12(4): 412-415.
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