高效制备霍乱弧菌菌影及其增强猪瘟疫苗免疫效果的研究

Production of Vibrio cholerae ghosts and its effect on enhancing immunity of classical swine fever vaccine

为评价霍乱弧菌菌影(VCG)对猪瘟活疫苗及猪瘟病毒E2蛋白抗原区(BC和AD区)亚单位疫苗免疫增强效果的作用,本实验首先利用含有裂解质粒的霍乱弧菌制备VCG,通过对细菌裂解前后进行平板计数及透射电镜检测VCG裂解效果,结果显示所制备的VCG裂解率高,且基本形成一个完整的细菌空壳。在确定最佳猪瘟活疫苗使用量后探究VCG对猪瘟活疫苗(CFS ST)免疫效果的影响,实验分别用VCG+CFS ST和CFS ST免疫家兔一次,设置对照组,并在免疫第42 d后攻毒。本研究还探究了VCG作为佐剂对猪瘟E2蛋白主要抗原区蛋白亚单位疫苗免疫效果的影响,实验分别用完全弗氏佐剂(CFA)和VCG联合E2蛋白主要抗原区蛋白,通过间隔两周的方式分4次免疫家兔,设置对照组,并在免疫第56 d后攻毒。通过阻断ELISA方法每周检测家兔血清的抗体阻断率,免疫后以500 RID50的猪瘟病毒弱毒株通过耳缘静脉注射方式对家兔攻毒。结果显示,50 RID50的猪瘟活疫苗组家兔血清抗体阻断率显著降低(p<0.001);VCG+CFS ST组家兔猪瘟抗体阻断率在免疫14 d后大部分时间点显著高于CFS ST组(p<0.05),并且攻毒后仅对照组出现定型热反应;CFA(E2)组和VCG(E2)组的家兔猪瘟抗体阻断率均显著达到较高水平(p<0.001),并且攻毒后仅对照组出现定型热反应。以上结果表明VCG可作为免疫佐剂增强猪瘟活疫苗的免疫效果;VCG联合猪瘟病毒E2蛋白主要抗原区亚单位疫苗在家兔中也具有较好的免疫效果。本研究为研发廉价高效的疫苗佐剂提供重要的实验依据。

To evaluate the enhancing efficacy of Vibrio cholerae ghost(VCG)on the classical swine fever vaccine,VCG was used as an adjuvant in combination with commercial classical swine fever(CSF)living cell vaccine or the BC and AD regions of CSFV E2 protein subunit for the vaccination of rabbits.VCG was prepared using one Vibrio cholerae that containing a lysis plasmid,which had a high lysis rate and forms a complete bacterial empty shell by plate counting evaluation and transmission electron microscopy observation.The effect of VCG on the immune efficacy of CSF living cell vaccine was evaluated by immunization of rabbits once using VCG+CFS ST,CFS ST,or PBS,respectively,and all rabbits were challenged ear vein with 500 RID50 of classical swine fever attenuated strain at 42 days post immunization(dpi).In addition,the effect of VCG as an adjuvant on the immune efficacy of CSFV E2 protein subunit vaccine was also investigated,rabbit were immunized four times with major regions of CSFV E2 protein subunit vaccine in combination with complete Freund's adjuvant(CFA)and VCG,or PBS,respectively,and all rabbits were challenged ear vein with 500 RID50 of classical swine fever attenuated strain at 56 dpi.The blocking rate of serum antibodies in rabbit was detected by blocking ELISA.The results showed that the blocking rate of rabbit serum antibodies in 50 RID50 group was increased significantly,and reached a high level at 42 dpi(p<0.001).The blocking rate of rabbit serum antibodies in VCG+CSF ST group was significantly higher than that of rabbit in CSF ST group after 14 dpi(p<0.05).In the CFA(E2)and VCG(E2)groups,the antibody blocking rate of rabbit reached a high level(p<0.001).Following viral challengence,there was no fever reaction in all vaccinated groups.In conclusion,VCG could serve as an effective adjuvant to enhance immune efficacy of classic swine fever living cell vaccine and CSFV E2 protein subunit vaccine in rabbits.This study is meaningful for the development of economic and efficient vaccine adjuvants.