重组禽流感病毒H7N9 H7-Re1株培养条件的优化

【背景】高致病性禽流感疫情的暴发造成了巨大的经济损失和环境卫生的破坏,现阶段疫苗接种仍是我国控制禽流感的主要措施之一,需要大量安全、高效和低成本的禽流感病毒疫苗。鸡胚法制备禽流感病毒疫苗的工艺存在原料来源受限、过程复杂、个体差异、培养周期长和不易放大培养等缺陷。而利用生物反应器大规模培养动物细胞生产病毒疫苗,不仅可以大幅度提高单位产量,实现高密度细胞和高病毒产率,同时可保证产品质量。目前我国用于禽流感防控的疫苗为重组禽流感病毒(H5+H7)二价灭活疫苗(H5N1 Re-8株+H7N9 H7-Re1株)。国内细胞全悬浮工艺生产禽流感灭活疫苗单罐产能最大为6 000 L,高病毒含量抗原的提供是生产高效疫苗的主要影响因素之一。【目的】为了能够提供稳定的、高效的生产抗原,开展种毒驯化试验。【方法】将重组禽流感病毒H7N9 H7-Re1株分别在MDCK细胞及悬浮MDCK细胞上增殖。在MDCK细胞上通过不同的病毒接种剂量、不同收获时间、不同TPCK-胰酶浓度的试验,确定了H7N9 H7-Re1株在MDCK细胞上最佳收获时间为64 h,最佳接毒剂量为0.008%或MOI为10~(-4),最佳TPCK-胰酶浓度为2μg·mL~(-1),根据确定的最佳培养条件连续传代,并对各代次病毒含量进行检测。【结果】在MDCK细胞上传至第5代时,HA可达1﹕256,每1 mL病毒含量达到10~(8.5)TCID50,每0.1 mL病毒含量达到10~(8.5)EID50,均高于其他代次。【结论】将第5代确定为MDCK细胞传代最佳代次,可考虑确定为生产用基础种毒代次。在悬浮MDCK细胞上对重组禽流感病毒传代进行了优化试验,确定了H7N9 H7-Re1株在悬浮MDCK细胞上最佳收获时间为48 h,最佳接毒剂量MOI为10~(-2),最佳TPCK-胰酶浓度为4—8μg·mL~(-1)。在实际疫苗生产过程中,可选择MDCK细胞或悬浮MDCK细胞来扩繁种毒。

Optimization of Cultivation Conditions for Reassortant Avian Influenza Virus H7N9 H7-Re1 Strain

【Background】Outbreaks of highly pathogenic avian influenza outbreak caused huge economic loss and damage the environment health, current vaccination remains one of the main measures to control avian influenza in China, which requires a lot of safety, high efficiency and low cost of the avian influenza vaccine. Due to the limitations of raw material sources, complex process, individual differences, long cultivation period and difficulty in amplifying culture, the process of preparing avian influenza vaccine by chicken embryo method is deficient. However, it is gradually becoming a trend to use bioreactor to produce virus vaccine on a large scale.It can not only increase the output of units, but also achieve high density cell and high virus yield, and ensure the quality of products. Currently, China’s vaccine for the prevention and control of avian influenza is the reassortant avian influenza virus (H5+H7) divalent inactivated vaccine (H5N1 Re-8 strain +H7N9 H7-Re1 strain). The production capacity of the single tank of avian influenza inactivated vaccine is the maximum of 6 000 L.The supply of high viral antigen is one of the main factors influencing the production of high efficiency vaccine.【Objective】In order to provide stable and efficient production antigens, a kind of domestication tests were carried out. 【Method】The reassortant avian influenza virus H7N9 H7-Re1 was proliferated on MDCK cells and suspended MDCK cells. Virus titers were tested by comparing different harvest times, viral inoculation doses and concentrations of TPCK on MDCK cells which the reassortant avian influenza virus H7N9 H7-Re1 strain were cultured in. The best harvest time was determined, 64 hours, dosage 0.008% or the MOI 10^-4 and TPCK-trypsin concentration 2 μg·ml^-1. According to the determined optimal culture conditions, the virus titer of each generation was tested. 【Result】The results showed that when virus were extended up to the fifth generation, HA reached 1:256 and the content of every 1 ml virus reached 10^8.5 TCID₅₀, and the content of every 0.1 ml virus reached 10^8.5 EID₅₀. Therefore, it can be determined that the 5th generation virus is the best generation for producing virus. 【Conclusion】The reassortant avian influenza virus was optimized and tested on the suspension MDCK cells. The best harvest time of the H7N9 H7-Re1 strain on suspension MDCK cells was 48 h, MOI 10^-4, and the best TPCK-trypsin concentration was 4-8 μg·ml^-1. In the actual production, MDCK cells or suspended MDCK cells can be selected to expand the virus.