整合猪瘟病毒囊膜糖蛋白假型鼠白血病病毒微量中和试验的建立和初步应用  

Establishment and Preliminary Application of Classical Swine Fever Virus Pseudovirions Microneutralization Assay

作　　者：周斌[1] 刘珂[1] 魏建超[2] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,南京210095 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《畜牧兽医学报》2011年第11期1570-1576,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(3100106230571376);南京农业大学青年科技创新基金(KJ08016);江苏高校优势学科建设工程资助项目

摘　　要：将构建的真核表达载体pcDNA-E0、pcDNA-E2和pcDNA-E012分别与MuLV假型病毒构建体系的2种骨架载体pHIT60和pHIT111经磷酸钙瞬时共转染293T细胞,48h后收集假病毒上清,超速离心后用抗CSFV多抗通过Western blot分析发现只有E012蛋白能够在假病毒颗粒表面表达,说明E012能够整合到假型病毒粒子表面。将此假病毒(MuLV-E012)感染SK6、PK-15、ST、BHK21、Vero、COS7、293T和CEF等8种细胞,48h后检测发现只有在SK6、PK-15和ST等猪源细胞中标记基因LacZ能有效表达,表明所构建的假病毒具有感染性,只能感染猪源细胞,进一步证实了猪瘟病毒对猪的单一嗜性,并且感染性与NH4Cl浓度存在极显著的线性负相关性,结果表明猪瘟病毒进入宿主细胞的过程有pH依赖性。将假病毒MuLV-E012代替强毒Shimen株与标准阴性、阳性血清和倍比稀释的待检血清混合后感染PK-15细胞,建立了微量中和试验,与全病毒微量中和试验进行了比较,结果表明所建立的方法能够代替强毒进行血清中和试验,检测临床血清的猪瘟病毒抗体中和效价。Three plasmids,namely pcDNA-E0/2/012,pHIT60（including the structural genes of MuLV） and pHIT111（including the retroviral genome,containing LacZ as a reporter） were co-transfected into HEK293T cells for the production of pseudotyped virions with E0/2/012 glycoproteins of classical swine fever virus（CSFV） Shimen strain.The retroviral supernatants were harvested at 48 hours post-transfection and used in western blot and infection assays.Western-blotting revealed only E012 could be expressed on the virions,indicated that the glycoprotein E012 was incorporated onto the retroviral virions.Infection test were performed on SK6,PK-15,ST,BHK21,Vero,COS7,HEK293T and CEF cells.The results showed that SK6,PK-15 and ST infected were LacZ positive,indicating viral entry,and revealed that the pseudtype virions of MuLV-E012 were infectious.To assess whether the CSFV pseudotyped virus entry is pH-dependent,PK-15 cells were treated with well-characterized lysosomotropic agent NH4Cl.Treatment with 30 mmol·L-1 NH4Cl caused 90% inhibition of infection by MuLV-E012 or MuLV-VSV G.These data indicated that the MuLV-E012＇s entry may be pH-dependent.The pseudotyped MuLV-E012 particles were used to develop an in vitro microneutralization assay that was both sensitive and specific for CSFV neutralizing antibody.Neutralization titers measured by this assay were highly parallel with those measured by the assay using live CSFV high virulence strain.Because the pseudotype assay does not require handling live CSFV virus,it is a useful tool to determine serum neutralizing titers during natural infection and the preclinical evaluation of candidate vaccines.

关 键 词：猪瘟病毒 囊膜糖蛋白 鼠白血病病毒 假病毒 感染性 微量中和试验 

分 类 号：S852.61[农业科学—基础兽医学]