猪乙型脑炎病毒在Vero细胞上的繁殖特性及其灭活疫苗的免疫原性研究

doi:10.16431/j.cnki.1671-7236.2018.12.031

摘要/Abstract

摘要：

试验旨在对猪乙型脑炎病毒（JEV，SA14-14-2株）在传代细胞上的繁殖培养特性及其制备的灭活疫苗免疫原性进行研究，确定猪JEV在细胞培养瓶中培养的关键技术参数及其灭活疫苗的免疫原性。以Vero细胞培养病毒，通过接种时间、接毒量、吸附时间、吸附温度、维持液pH、维持培养温度及培养时间7个条件的优化，将繁殖的病毒液冻融一次，采用病毒蚀斑数测定方法测定病毒滴度。按照优化好的条件繁殖一批毒液，经β-丙内酯灭活，与双相佐剂混合，制备成猪乙型脑炎灭活疫苗。两次免疫（间隔14 d）接种乙型脑炎抗体阴性仔猪，首次免疫前（0 d）、免疫后第7、14、21、28、35、42天采集血清，检测血清中和抗体。二免后第28天进行乙型脑炎P3强毒的攻击，攻毒前（0 d）、攻毒后第1、2、3、5、7、9天采集血浆，攻毒后第14天剖杀免疫猪，采集脑组织，检测血浆和脑组织中JEV。结果显示，用细胞培养瓶进行培养，将Vero细胞培养至48 h进行病毒接种，接种量为1 000 PFU/mL，病毒吸附温度为37℃，吸附时间为90 min，吸附后用pH 7.6~8.8维持液继续培养，培养温度为35℃，培养96 h后收获毒液，冻融一次，可获得较高滴度的病毒。仔猪免疫制备的灭活疫苗后血清抗体水平迅速升高，血浆和脑组织中均未检测出JEV，免疫组试验猪能抵抗强毒攻击，可获得有效免疫保护。本试验结果为猪乙型脑炎疫苗的生产提供了参考依据。

关键词: 乙型脑炎病毒(JEV); Vero细胞; 繁殖特性; 病毒滴度; 灭活疫苗; 免疫原性

Abstract:

This study was aimed to investigate the reproductive characteristics of swine Japanese encephalitis virus (SA14-14-2 strains) in Vero cell and the immunogenicity of its inactivated vaccine.The swine Japanese encephalitis virus was cultured in Vero cell.The proliferation technology of swine Japanese encephalitis virus in Vero cell line was studied by the selection and optimization of the following seven culture conditions,including Vero cell incubation time,the amount of inoculated virus,adsorption time,adsorption temperature,pH of maintenance fluid,maintain culturing temperature and culture time.After one time of freezing and thawing,the virus titer was determined by means of viral plaque number determination.The inactivated vaccine of swine Japanese encephalitis virus was prepared by reproducing a batch of viruses under optimized conditions and inactivating it by means of inactivation of β-propiolactone and mixed with bidirectional adjuvant.Two immunizations (interval 14 d) were given to piglets with negative antibody to encephalitis,and the serum was collected before (0 d) and after 7,14,21,28,35 and 42 d for the first immunization,and serum neutralization antibodies were detected.We attacked piglets using the encephalitis P3 strain at the 28th after two immunizations,and the plasma was collected before (0 d) and after 1,2,3,5,7 and 9 d;14 d after the attack,piglets were dissected and brain tissues were collected,and detecting the swine Japanese encephalitis virus in plasma and brain tissue.The results showed that the cells were cultured in a cell culture flask,and the Vero cells were cultured for 48 h for virus inoculation,the inoculum size was 1 000 PFU/mL,the virus adsorption temperature was 37℃,the adsorption time was 90 min,and pH was 7.6 to 8.8 after adsorption.The maintenance solution was continued to be cultured at a temperature of 35℃.After 96 h of culture,the venom was harvested and frozen and thawed to obtain a higher titer.The serum antibody level of the inactivated vaccine prepared by immunization of piglets increased rapidly.No Japanese encephalitis virus was detected in plasma or brain tissue.The test piglets of immunized group could resist strong poisoning and obtain an effective immune protection.This results provided a basis for the production of swine encephalitis vaccine.

Key words: Japanese encephalitis virus (JEV); Vero cells; reproductive characteristics; virus titer; inactivated vaccine; immunogenicity

中图分类号:

S852.65

韩伟, 周建民, 郝霖雨, 潘文, 潘京学. 猪乙型脑炎病毒在Vero细胞上的繁殖特性及其灭活疫苗的免疫原性研究[J]. 《中国畜牧兽医》, 2018, 45(12): 3572-3578.

HAN Wei, ZHOU Jianmin, HAO Linyu, PAN Wen, PAN Jingxue. Study on the Reproductive Characteristics of Swine Japanese Encephalitis Virus in Vero Cells and the Immunogenicity of Its Inactivated Vaccine[J]. , 2018, 45(12): 3572-3578.

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