Expression of RSV F protein fragment and its application
AIM:To establish rapid,sensitive,specific,cheap serological detection methods of human respiratory syncytial virus.METHODS:The viruses were isolated from nasopharyngeal secretions which collected from patient in BeiJing Children's hospital.One fragment of F gene(F1-1,AA 137-363)was amplified by RT-PCR,since this region contains the major neutralization sites.The PCR product was cloned into pMD18-T vector and was identified by PCR,then the target gene was subcloned into pET-32a expression vector and was identified by restriction analysis and sequencing.F1-1 was efficiently expressed in E.coli BL21 and purified by nickel HiTrap chelating metal affinity column.The serum levels of IgG antibodies to F antigen were measured in 91 serum samples of suspected RSV from BeiJing Children's hospital using indirect ELISA against F1-1 proteins of HRSV.RESULTS:The size of the RT-PCR product was 678 bp.The sequence of insert was correct after examined by restriction enzyme digestion and sequencing.Western Blotting indicated that the RSV F1-1 protein had specific antigenicity.No significant difference was found when compared our method with commercial detection kits.CONCLUSION:The recombinant RSV F protein fragment offers an efficient way for serological diagnosis.
human respiratory syncytial virusfusion(F)geneprokaryotic expression
NETL
NSTL
万方数据
维普
