摘要
为了提高猪细小病毒可溶性VP2蛋白表达并研制病毒样颗粒(virus-like particles,VLPs)疫苗,试验经密码子优化后,合成PPV VP2基因序列,构建pET30a-PPV-VP2重组表达质粒,并与pKJE7、pGro7、pTf163种分子伴侣共同转化至E.coli BL21(DE3)感受态细胞中,经诱导后分析其可溶性表达水平并优化诱导条件,进行SDS-PAGE及Western-blot鉴定。通过Ni-NTA亲和层析纯化,透析除去咪唑进行体外自组装,通过动态光散射和透射电镜观察VLPs粒径和形态。用制备的PPV VLPs疫苗采用肌肉注射免疫和鼻滴免疫昆明鼠评价其免疫原性。结果表明:当pET30a-PPV-VP2与pTf16共表达时,以0.1 mmol/L IPTG、2 g/L l-阿拉伯糖、30℃培养16 h为诱导条件,VP2蛋白在大肠杆菌的可溶性表达最高。VP2蛋白经纯化后,可自组装成直径约为23.52 nm的PPV VLPs,其血凝效价为1∶2~9。表达的VLPs制备成疫苗,通过肌肉注射方式免疫昆明鼠,HI效价极显著高于猪细小病毒灭活疫苗(P<0.001),诱导小鼠产生较高水平的抗体(IgG1、IgG2a、IgG2b、IgG3)和细胞因子[γ干扰素(IFN-γ)和白细胞介素-4(IL-4)]。鼻滴免疫VLPs可以诱导产生高水平的分泌型免疫球蛋白(sIgA),VLPs无明显的副作用,具有一定的安全性。说明制备的PPV VLPs具有较好的免疫原性,诱导昆明鼠产生细胞免疫、体液免疫及平衡的Th1/Th2免疫应答,鼻滴免疫VLPs疫苗时无需佐剂辅助可诱导产生高效的黏膜免疫应答。
In order to improve the soluble VP2 protein expression of Porcine parvovirus(PPV)and to develop a vaccine with virus-like particles(VLPs),in the experiment,after codon optimization,the PPV VP2 gene sequence was synthesized and pET30a-PPV-VP2 recombinant expression plasmid was constructed,and it was co-transformed with three molecular chaperones,pKJE7,pGro7 and pTf16,into E.coli BL21(DE3)competent cells.After induction,the soluble expression level was analyzed and the induction conditions were optimized,and SDS-PAGE and Western-blot were performed for identification.Purified by Ni-NTA affinity chromatography,imidazole was removed by dialysis for in vitro self-assembly,and the particle size and morphology of VLPs were observed by dynamic light scattering and transmission electron microscopy.The immunogenicity of the prepared PPV VLPs vaccine was evaluated by using intramuscular immunization and nasal drip immunization of Kunming rats.The results showed that when pET30a-PPV-VP2 was co-expressed with pTf16,the soluble expression of VP2 protein in E.coli was the highest when 0.1 mmol/L IPTG,2 g/L l-arabinose,and 30℃incubation for 16 h were used as the induction conditions.The VP2 protein was purified to self-assemble into PPV VLPs with a diameter of approximately 23.52 nm and a hemagglutination potency of 1∶2~9.The expressed VLPs were prepared as a vaccine and immunized by intramuscular injection in Kunming mice;the HI titer was highly significantly higher than that of the inactivated Porcine parvovirus vaccine(P<0.001),and the mice were induced to produce higher levels of antibodies(IgG1,IgG2a,IgG2b,IgG3)and cytokines(γ-interferon[IFN-γ]and interleukin-4[IL-4]).Nasal droplet immunization with VLPs induced the production of high levels of secretory immunoglobulin(sIgA),and the VLPs had no significant side effects and were safe.The results indicated that the prepared PPV VLPs had good immunogenicity,induced cellular and humoral immunity,balanced Th1/Th2 immune response and efficient mucosal immune response in Kun
作者
吕兆伟
刘冰
李耀政
吴惠宇
徐雨昕
王丹红
吴丛梅
殷玉和
LYU Zhaowei;LIU Bing;LI Yaozheng;WU Huiyu;XU Yuxin;WANG Danhong;WU Congmei;YIN Yuhe(College of Chemistry and Life Science,Changchun University of Technology,Changchun 130012,China;Jilin Five-Star Animal Health Co.,Ltd.,Changchun 130012,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2024年第14期102-110,128,共10页
Heilongjiang Animal Science And veterinary Medicine
基金
吉林省科技发展计划项目(20200404195YY)。
关键词
猪细小病毒
分子伴侣
VP2蛋白
病毒样颗粒
免疫原性
Porcine parvovirus
molecular chaperone
VP2 protein
virus-like particle
immunogenicity
