甲型副伤寒杆菌重组蛋白OmpW的表达与鉴定

Prokaryotic expression and identification of recombinant outer membrane OmpW fromSalmonella paratyphi A

目的构建甲型副伤寒杆菌外膜蛋白基因ompW的原核表达系统,确定其重组表达产物rOmpW免疫原性,了解甲型副伤寒杆菌临床菌株ompW基因携带率。方法采用PCR和T-A克隆法从甲型副伤寒杆菌临床株JH01中获得ompW基因克隆并构建其原核表达系统,重组蛋白纯化后用SDS-PAGE及Western-Blotting分析。采用PCR检测95株甲型副伤寒杆菌临床菌株ompW基因携带率。结果与报道的相关序列比较,所克隆的ompW基因核苷酸和氨基酸序列相似性均为100%。rOmpW免疫家兔可产生抗体并能与甲型副伤寒杆菌全菌抗血清产生阳性Western杂交信号。所有甲型副伤寒杆菌菌株均携带ompW基因。结论构建甲型副伤寒杆菌外膜蛋白基因ompW的原核表达系统,证明了ompW是存在于甲型副伤寒杆菌的序列保守、分布广泛的外膜蛋白基因,为进一步研究该蛋白作为多价甲型副伤寒杆菌基因工程疫苗候选抗原奠定基础。

The purpose of this study was to generate a prokaryotic expression system of Salmonella paratyphi A ompW gene that encoding an outer membrane protein（OMP）,determine immunogenicity of the recombinant expressed product rOmpW,and carry out frequencies of the ompW genes in isolates of S.paratyphi A.A clone ompW gene was obtained from a clinical S.paratyphi A strain JH01 by using PCR and T-A cloning method,and then a prokaryotic expression system of the gene clone was generated.SDS-PAGE and Western-Blotting were applied to examine the expression and yield of rOmpW.The carrying rates of ompW genes in 95 S.paratyphi A isolates were detected by PCR.Results exhibited that all the cloned ompW genes had 100% nucleotide and putative amino acid sequence identities compared to the reported sequencing data.The rOmpW could efficiently induce rabbits to produce specific antibody and present positive Western hybridization signals with S.paratyphi A antiserum.All the tested S.paratyphi A strains had ompW gene as well as expressed OmpW protein.In conclusion＇ the S.paratyphi A ompW gene were successfully amplified and cloned.OmpW is an OMP antigen gene of S.paratyphi A with conserved sequence and extensive distribution.This OMP could be used as one of the candidate antigens for developing multiple-valence genetic engineering vaccine of S.paratyphi A.