SARS-CoV假病毒中和试验技术的建立及评价  

Development and Application of a Safe SARS-CoV Neutralization Assay Based on Lentiviral Vectors Pseudotyped with SARS-CoV Spike Protein

作　　者：闫克夏[1] 谭文杰[1] 张相民[1] 王慧娟[1] 李岩 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]加拿大国家微生物实验室

出　　处：《病毒学报》2007年第6期440-446,共7页Chinese Journal of Virology

基　　金：国家863计划2007AA02Z464;病毒病预防控制所2005年SARS疾控专项经费

摘　　要：为避免传统的SARS病毒中和试验需要操作活毒而存在的生物安全隐患,建立了基于假病毒系统、操作较安全的SARS中和试验技术平台。本研究应用高效表达SARS-CoV S(密码子优化的全长S蛋白,简称S)的真核表达载体(pVRC8304),与HIV慢病毒包装质粒(p CMV△8.2)及转移质粒(pHR′CMV EGFP)3个质粒载体系统共同转染人胚肾细胞293T,包装了SARS假病毒;通过SARS假病毒感染的RD-A细胞中标记基因EGFP表达的分析,确定SARS假病毒能有效进入细胞,建立了可在BSL-2级实验室操作的SARS病毒中和试验技术平台。用该技术平台对不同免疫血清进行了中和抗体分析,并比较了基于假病毒和基于SARS活病毒的中和试验效果。结果显示:SARS假病毒和SARS活病毒两个中和试验系统获得中和抗体滴度变化趋势一致,表明本研究构建的SARS假病毒可替代SARS活病毒用于建立操作上安全的SARS病毒中和试验技术平台。The severe acute respiratory syndrome-associated coronavirus （SARS-CoV） spike protein （S） is a major target for neutralizing antibody. To develop and apply a safe neutralization assay for SARS-CoV, lentiviral SARS-CoV S pseudotypes had been constructed based on a three plasmid system , which contained pVRC8304（ harboring codon optimized full-length SARS-CoV S protein） , pCMVA8.2 （HIV-1 gag/pol construct） and pHR＇CMV EGFP（the green fluorescent protein reporter construct） . The pseudotyped lentiviral particles were used to develop an in vitro microneutralization assay that was both sensitive and specific for SARS-CoV neutralizing antibody. We used this assay to determine the titers of the neutralizing antibodies （Nabs） in serum samples from mice immunized with various rVVs expressing different S fragments of SARS-CoV. The serum antibodies derived from S and various segments of S1 region neutralized SARS-CoV in vitro. No cross-neutralization occurred with the goat antiserum prepared with inactivated HCoV-OC43 or HCoV-229E. Neutralization titers measured by this assay were highly parallel with those measured by the assay using live SARS-CoV. Because the pseudotype assay does not require handling live SARS virus, it is a useful tool to determine serum neutralizing titers during natural infection and the preclinical evaluation of candidate vaccines.

关 键 词：SARS-COV S蛋白 假病毒 中和试验 

分 类 号：R373.1[医药卫生—病原生物学]