| 吴德平1 焦留宏1 王加林1 杨文娟1 陈金拳1 王猛1 王颖2.立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析[J].,2011,11(6):1063-1067 |
| 立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析 |
| Cloning, Expression and Immunogenicity of Outer Membrane Protein HGene Fragment of Rickettsia Rickettsii in E.coli |
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| DOI： |
| 中文关键词: 立氏立克次体 基因重组 外膜蛋白H |
| 英文关键词: Rickettsia rickettsii gene recombinant outer membrane protein H |
| 基金项目: |
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| 中文摘要: |
| 目的：克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H 基因(ompH)片段并对其进行免疫原性分析。方法：采用PCR
技术从立氏立克次体基因组中扩增ompH 基因片段，将该基因片段与原核表达载体pET32a 连接，构建重组原核表达质粒
pET32a/ompH ;将pET32a/ompH 转入大肠杆菌细胞内，用IPTG 诱导转化大肠杆菌表达目的基因。结果：获得长为327bp 的ompH
基因片段，SDS-PAGE 分析发现pET32a/ompH 转化菌表达了大小约27kDa 蛋白, 该蛋白与立氏立克次体免疫豚鼠血清及斑点热
患者血清在免疫印迹分析中呈阳性反应, 经该重组蛋白免疫血清中和后的立氏立克次体感染VERO 活力减低。结论：
pET32a/ompH 转化的大肠杆菌表达了ompH 基因片段，所产生的重组蛋白具有良好的免疫反应性及保护性。 |
| 英文摘要: |
| Objective: To express the gene encoding outer membrane protein H (OmpH) of Rickettsia rickettsii(R.r) in E.coli and
to investigate the immunogenicity. Methods: The gene fragment encoding the outer membrane protein H (OmpH) was amplified from the
genomic DNA of R .r by PCR, and was transferred into the prokaryotic expression vector pET32a to construct the recombinant plasmid
pET32a/omph. The recombinant plasmid were transformed into the E. coli cells and the recombinant gene were induced to express protein
by IPTG. The recombinant protein was analyzes by immunoblottingg assay. Results: A ompH gene fragment with length of 486 bp
was cloned,The recombinant protein of approximately 27kDa could recognized by sera of the infected guinea pigs and sera from patients.
Before being added to suspended VERO cells to assess their infectivity by fluorescent quantitative PCR assays, hyperimmune antirickettsial
sera, prepared in mouse by injections of the recombinant protein and R.r in growth medium were incubated in serum at room temperature
for 30 min (pretreatment). It was concluded that immune serum may inhibit rickettsial infection. Conclusions: E coli cells transformed
by plasmid pET32a/ompH can express a 29 kDa recombinant protein and this protein may be the major antigens of R. r capable
to induce specific immune responses and may be used as the new candidate to develop diagnostic reagents or to prepare the subunit vaccine
for spotted fever. |
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