摘要
[目的]验证利用革兰氏阳性增强基质颗粒(GEM)浓缩纯化口蹄疫病毒(FMDV)抗原的可行性。[方法]将含有3个自溶素基序的锚钩蛋白基因(PA)与针对O型口蹄疫病毒特异性纳米抗体基因(VHH)串联克隆入原核表达载体p ET-32a(+)中,构建原核表达载体p ET-VPA,将其转化大肠杆菌宿主菌BL21(DE3),表达获得融合重组蛋白VPA。先将GEM颗粒与重组蛋白VPA在37℃孵育30 min,离心,取沉淀用适量的FMDV抗原液重悬,再经一步低速离心,获得沉淀即为浓缩纯化的口蹄疫病毒抗原。利用SDS-PAGE、Western-blot、蛋白含量测定及146S测定等方法对浓缩纯化方法的可行性进行鉴定,并通过动物免疫试验初步验证浓缩纯化后抗原的免疫原性。[结果]VPA融合蛋白能够在大肠杆菌中部分可溶性表达。作为接头蛋白,VPA既能与GEM颗粒结合,又能与FMDV抗原结合。SDS-PAGE与Western-blot结果表明:FMDV通过GEM法得到有效纯化;146S测定结果表明FMDV的回收效率达到99%;总蛋白含量测定结果显示口蹄疫抗原杂蛋白去除率达到90%;动物免疫试验结果显示GEM技术纯化后的FMDV具有良好的免疫原性。[结论]利用GEM技术浓缩纯化口蹄疫病毒抗原是可行的。
[ Objectives] The purpose of this study is to verify the feasibility of purification of type O foot and mouth disease virus （FMDV） inactivated antigen with Gram-positive bacterial enhancer matrix （GEM）technology. [ Methods ] A dual functional molecular consisting of anchor protein gene and FMDV specific single domain antibody gene of type O FMDV were expressed in Escherichia coli,and the recombinant protein was named as VPA. The sedimentation named GEM-VPA was obtained with centrifugation following mixing the recombinant protein with GEM particles, and incubated at 37 ~C for 30 min ; GEM-VPA was mixed with FMDV antigen, and then the sedimentation was collected with low speed centrifugation after incubated at 37 ~C for another 1 h, which was the purified antigen named GEM-FMDV. The feasibility for the method was evaluated with SDS-PAGE, Western-blot, protein quantification and 146S detection, and the immunogenicity for purified FMDV antigen was estimated with animal test. [ Results ] The dual functional protein designed in this study could be partly soluble when expressed in Escherichia coll. Moreever, this recombinant protein could bind with GEM particles and inactivated FMDV antigen at the same time. SDS-PAGE and Western-blot proved that the purified FMDV antigen existed in the sedimentation after centrifugation. Results from 146S determination indicated that the FMDV antigen recovery in this study could be above 99%, and the removal efficiency of the heterologous proteins from FMDV antigen was higher than 90%. More importantly, animals injected with the purified antigen developed more vigorous immune response and lesser side effect. [ Conclusions] It is feasible for FMDV antigen concentration and purification with GEM technology.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2018年第1期147-153,共7页
Journal of Nanjing Agricultural University
基金
江苏省农业自主创新专项资金项目(CX(15)1026)
关键词
GEM技术
口蹄疫病毒
抗原纯化
GEM technology
foot and mouth disease virus
antigen purification
