摘要
为研制新型、可用于区分野毒感染、助力猪瘟净化的亚单位疫苗,本研究参考了近年来国内猪瘟病毒(CSFV)流行株的E2基因序列,并根据昆虫细胞密码子偏嗜性对其进行优化、合成后,克隆至转移载体中,构建了重组质粒,随后转化至含有穿梭质粒的大肠杆菌DH10Bac,制备了重组杆粒并转染至SF9细胞,获得了表达E2基因的重组杆状病毒。间接免疫荧光试验和western blot检测结果显示该杆状病毒感染昆虫细胞后可正确表达E2蛋白(Bac-E2)。将Bac-E2(50μg/头份)与猪瘟兔化弱毒疫苗[HCLV,7500半数兔体感染剂量(RID50)/头份]分别免疫猪瘟抗体阴性仔猪,3周后用相同的剂量加强免疫。首免后5周Bac-E2组猪CSFV中和抗体均不低于11024,显著高于HCLV组(p<0.01);首免后35 d,利用105.0MLD(最小致死剂量)的CSFV石门强毒经耳后颈部肌肉注射攻毒后,Bac-E2和HCLV组猪的保护率均为100%,阴性对照组猪全部发病、死亡;采用荧光定量PCR检测各组猪口腔和肛门拭子中CSFV的排毒情况,结果显示Bac-E2和HCLV组猪攻毒后0~16 d的咽拭子及肛拭子中均未检测到CSFV核酸。阴性对照组猪攻毒后第4 d至濒死前均可在其拭子中检出高拷贝CSFV核酸。本研究为Bac-E2蛋白作为猪瘟亚单位疫苗的候选抗原蛋白奠定了研究基础。
In order to develop a novel subunit vaccine of classical swine fever,the E2 gene sequence of pandemic CSFV strains in recent years was optimized and synthesized according to the codon biasof insect cells.The optimized E2 gene was cloned into pFastBac1 vector to construct the recombinant plasmid.After transforming DH10Bac competent cells,the recombinant Bacmid-E2 was confirmed by PCR and transfected into SF9 cells to obtain recombinant baculovirus.The production of E2 proteinin recombinant baculovirus was validated by indirect immunofluorescence assay and western blot assay.In addition,CSFV antibody-negative piglets were immunized with Bac-E2 protein(50μg/dose)and hog cholera lapinized virus(HCLV)respectively,and boosted with same dose after three weeks.The CSFV neutralizing antibody titers of Bac-E2 group were noless than 1:1024 after 35 days of first immunization,which was significantly higher than HCLV group.Piglets in Bac-E2 group and HCLV group were 100%protected from CSFV(Shimen strain)challenge.In contrast,all negative control pigs displayed severe CSF disease with100%mortality by 12 days post challenge.Analysis of pharyngeal andanal swabs by quantitative real time RT-PCR showed that none of the Bac-E2 and HCLV vaccinated pigs was positive for CSFV from 0 to 16 dpc,while all negative control pigs were positive for CSFV from 4 dpc to death.These results indicated that the Bac-E2 protein could be a promising candidate subunit vaccine against CSFV.
作者
王遵宝
贺笋
李俊辉
刘宏
豆智华
郑侃
李森江
寇春
WANG Zun-bao;HE Sun;LI Jun-hui;LIU Hong;DOU Zhi-hua;ZHEN Kan;LI Sen-jiang;KOU Chun(Tecon Bio-technology Co.,Ltd.,Urumqi 830032,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第4期383-388,共6页
Chinese Journal of Preventive Veterinary Medicine
关键词
猪瘟病毒
昆虫细胞
杆状病毒
免疫原性
classical swine fever virus
insect cell
baculovirus
immunogenicity
作者简介
第一作者:王遵宝(1984-),男,山西大同人,中级兽医师,主要从事兽用疫苗研发;第一作者:贺笋(1980-),男,新疆阿克苏人,主要从事兽用疫苗研发;通信作者:李俊辉,E-mail:lijh0616@163.com
