猪轮状病毒油乳剂灭活疫苗的制备及免疫性试验

发布时间：2018-06-30 22:16

本文选题：猪轮状病毒 + 灭活疫苗　；参考：《东北农业大学》2009年硕士论文

【摘要】： 猪轮状病毒(Porcine Rotavirus, PRV)属呼肠病毒科轮状病毒属成员,是引起仔猪病毒性腹泻的重要病原之一,该病在世界范围内普遍存在,给世界各地的畜牧业造成严重的损失。该病主要感染幼畜,1~10日龄仔猪感染后发病率可超过80%,死亡率高达50%~100%。主要症状为严重腹泻,部分病例因严重脱水、酸碱平衡失调并且继发感染而死亡。目前还没有治疗轮状病毒腹泻的特效药物。接种疫苗来预防轮状病毒感染显得尤为重要。因此,世界卫生组织多次呼吁各国科学家研制开发有效的轮状病毒疫苗,并将其列为最优先发展的疫苗项目之一。本实验制备了猪轮状病毒油乳剂甲醛和BEI灭活疫苗,并以家兔为模型动物,肌肉接种后进行了疫苗免疫原性的测定。将猪轮状病毒分离株JL94株在恒河猴肾细胞系(MA-104)细胞系上传代,通过电镜技术、RT-PCR、序列测定和间接免疫荧光对猪轮状病毒毒种进行了鉴定,证明该病毒为典型的猪轮状病毒。将该病毒在MA-104上传代增殖,当病毒传至第58代,滴度达到108.25TCID50/ml,制备出基础种毒。并对种毒进行了细菌和支原体检测,检测结果均为阴性。将其扩大培养,制备了生产种毒,作为制备疫苗的抗原液。将该抗原液分别用甲醛和BEI灭活,制备油佐剂疫苗,然后肌肉免疫接种家兔。将家兔随机分为3组,每组3只,分别命名为甲醛组、BEI一免组和BEI二免组。该疫苗注射家兔后均未引起动物的发病与死亡,注射部位未见明显的病理变化,免疫家兔无任何不良反应,精神、食欲良好,证明了该疫苗安全性良好。免疫后,每隔一周耳静脉取血,分离血清,用间接ELISA法和病毒在细胞水平上的中和试验检测血清中特异性IgG抗体。通过间接ELISA检测表明,甲醛组家兔在一免时抗体水平升高不明显,未检测出抗体效价,在免后2w进行加强免疫,加强免疫后1w的抗体水平明显升高,达到峰值为1:9600,接着进入了下降阶段,14w至17w,ELISA抗体效价维持在1:1200;而BEI一免组和二免组家兔血清中IgG抗体效价均有明显的上升,峰值均为1:9600,至免疫后的21w一免组的抗体效价仍能维持在1:1000,二免组为1:4000;中和试验结果表明,甲醛组首次免疫后中和抗体效价升高缓慢,第3w加强免疫后中和抗体效价有明显的升高趋势,峰值为1:969,后逐渐降低,检测到15w降低到1:255;而BEI一免组和二免组家兔,抗体效价变化规律基本一致,均在第3w抗体效价明显升高,一免组和二免组中和抗体效价峰值均为1:1800,于免疫后14w抗体效价开始缓慢下降,免疫后21w一免组的抗体效价降低为1:119,二免组为1:230。两种检测方法均显示,猪轮状病毒油乳剂BEI灭活疫苗比猪轮状病毒油乳剂甲醛灭活疫苗免疫家兔后所产生的体液抗体效价高且持续时间长,且对家兔进行一次免疫与两次免疫效果相当,因此可得猪轮状病毒油佐剂BEI灭活疫苗的免疫效果好,且用其对动物进行一次免疫即可。
[Abstract]:Porcine Rotavirus (PRV), a member of rotavirus genus of the family Reovirus, is one of the most important pathogens causing viral diarrhea in piglets. The incidence of the disease can exceed 80% and the mortality is as high as 50% and 100% after infection in 10 days old piglets. The main symptoms were severe diarrhea, and some died of severe dehydration, acid-base imbalance and secondary infection. There is no specific drug available to treat rotavirus diarrhea. Vaccination against rotavirus infection is particularly important. Therefore, the World Health Organization has repeatedly appealed to scientists in various countries to develop an effective rotavirus vaccine and make it one of the top priority vaccine projects. The inactivated vaccine of porcine rotavirus oil emulsion formaldehyde and BEI was prepared in this experiment. The immunogenicity of the vaccine was determined after intramuscular inoculation in rabbits. Porcine rotavirus strain JL94 was isolated from rhesus monkey kidney cell line (MA-104). The strain of porcine rotavirus strain JL94 was identified by electron microscope technique with RT-PCR, sequencing and indirect immunofluorescence. It was proved that the virus is a typical porcine rotavirus. When the virus was transmitted to the 58th generation, the titer of the virus reached 108.25TCID 50 / ml, and the base virus was prepared. Bacteria and mycoplasma were detected and the results were negative. It was expanded to produce the seed virus, which was used as the antiviral solution for the preparation of vaccine. The antigenic solution was inactivated by formaldehyde and BEI respectively to prepare oil adjuvant vaccine and then immunized rabbits intramuscularly. Rabbits were randomly divided into three groups with 3 rabbits in each group. There were no pathological changes in the injection site, no adverse reactions, good spirit and good appetite after injection of the vaccine, which proved that the vaccine was safe. After immunization, blood was collected from ear vein every other week and serum was isolated. Specific IgG antibody in serum was detected by indirect Elisa and virus neutralization test at the cell level. The results of indirect Elisa showed that the level of antibody in formaldehyde group was not significantly increased at the first immunity, but the antibody titer was not detected. The level of antibody was significantly increased at 2 weeks after immunization and 1 week after immunization. The peak value of Elisa antibody was 1: 9600, and the titer of Elisa antibody was maintained at 1: 1200 from 14w to 17w.The titer of IgG antibody in rabbit serum of BEI group and second immunity group increased obviously. The peak value was 1: 9600, and the titer of neutralizing antibody was maintained at 1: 1000 in the first immunized group and 1: 4000in the second immunity group at 21w after immunization. The neutralizing antibody titer of formaldehyde group increased slowly after the first immunization. The titer of neutralizing antibody increased obviously at the third week after immunization, the peak value was 1: 969, then decreased gradually, and the antibody titer decreased to 1: 255 at 15w, but the changes of antibody titer were basically the same in the BEI group and the second immunity group. The titer of neutralizing antibody in the first and second immunized groups was 1: 1800, and the antibody titer began to decrease slowly at the 14th week after immunization. The titer of the first and second immunized groups decreased to 1: 119and 1: 230respectively at the third week after immunization. Both methods showed that the humoral antibody titers of rabbits immunized with porcine rotavirus oil emulsion BEI inactivated vaccine were higher and lasted longer than that of porcine rotavirus oil emulsion formaldehyde inactivated vaccine. The inactivated vaccine of porcine rotavirus oil adjuvant BEI can be obtained, and it can be used to immunize animals once.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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