畜牧兽医学报2011，Vol.42Issue(11)：1570-1576,7.
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
摘要
Abstract
Three plasmids, namely pcDNA-E0/2/012, pHIT60 (including the structural genes of MuLV) and pHITlll (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 glyco-proteins 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-de-pendent, PK-15 cells were treated with well-characterized lysosomotropic agent NH4C1. Treatment with 30 mmol · L-1 NH4C1 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 pseudo-typed 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Key words
CSFV/E012/murine leukemia virus/pseudotype virus/infectivity/microneutralization assay分类
农业科技引用本文复制引用
周斌,刘珂,魏建超,陈溥言..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[J].畜牧兽医学报,2011,42(11):1570-1576,7.基金项目
国家自然科学基金(31001062 （）
30571376) （）
南京农业大学青年科技创新基金(KJ08016) （KJ08016）
江苏高校优势学科建设工程资助项目 （）
