淋病奈瑟菌表面蛋白A克隆、表达和免疫原性鉴定(1)
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[摘要] 目的 克隆并构建淋病奈瑟菌表面蛋白A(nspA)基因原核表达系统，并对重组产物rNspA进行免疫原性鉴定。 方法 采用PCR扩增nspA基因，构建nspA基因原核表达系统。SDS-PAGE检测目的重组蛋白rNspA表达情况，Ni-NTA亲和层析法提纯rNspA，免疫双扩散试验及Western Blot鉴定rNspA的免疫原性。 结果 克隆的nspA基因与GenBank中相关序列相似性为100%，rNspA表达量为细菌总蛋白的40.1%，提纯的rNspA在SDS-PAGE后显示为单一的蛋白条带。rNspA抗血清免疫双扩散效价为1∶4，rNspA抗血清能有效识别rNspA。 结论 所构建的nspA基因原核表达系统能高效表达rNspA，表达的产物有良好的免疫原性，为淋病奈瑟菌免疫疫苗的研制奠定基础。
[关键词] 淋病奈瑟菌；nspA基因；克隆/重组表达；免疫原性鉴定
[中图分类号] R759.2 [文献标识码] A [文章编号] 1673-9701(2012)03-0009-02
Construction and prokaryotic expression of Neisseria gonorrhoeae nspA gene and immunological identification of the expressed product
DU Peng WU Senlin ZHONG Yawen SUN Aihua
Zhejiang Medical College, Hangzhou 310053, China
[Abstract] Objective To clone nspA gene of N.gonorrhoeae and then construct its prokaryotic expression system, and to identify the immunogenicity of its product. Methods nspA gene was amplified by PCR and its prokaryotic expression system was then constructed. SDS-PAGE was used to measure the expression of target recombinant protein rNspA, Ni-NTA affinity chromatography was applied to extract rNspA. Double immunodiffusion and Western Blot assay were applied to indentify immunogenicity of rNspA. Results Sequence similarity of the cloned nspA gene was 100% compared to the corresponding sequence in GenBank. The expression output of rNspA was approximately 40.1% of the total bacterial proteins. The extracted rNspA showed a single protein band in gel after SDS-PAGE. The immunodiffusion titer of rabbit antiserum against rNspA was 1∶4. The rNspA antiserum was able to recognize rNspA. Conclusion The constructed prokaryotic expression system enable express rNspA with high efficiency, and can be used as a candidate antigen for developing genetic engineering vaccine.
[Key words] Neisseria gonorrhoeae; NspA Gene; Clone/recombinant expression; Immunogenicity/identification
淋病奈瑟菌感染引起的淋病发病率目前居于我国各类性传播疾病首位[1]。淋病奈瑟菌感染后可产生特异性免疫力，但维持时间短导致重复感染。研究认为淋病奈瑟菌免疫逃逸机制可能与该菌外膜蛋白抗原多态性相关[2] ......
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