表面表达幽门螺杆菌中性粒细胞激活蛋白枯草芽孢的免疫原性

Immunogenicity of Bacillus subtilis spores surface expressing Helicobacter pylori neutrophil activating protein

目的 构建表面表达幽门螺杆菌中性粒细胞激活蛋白（NAP）的重组枯草芽孢,并鉴定其免疫原性.方法 以构建的pGEX-4T-1-NAP质粒为模板,采用特异性引物扩增幽门螺杆菌NAP基因,与构建的以枯草芽孢外衣蛋白Cotc融合表达的质粒Pus186-Cotc连接,转化枯草杆菌WB600,提取质粒进行酶切、测序鉴定.应用营养耗竭法诱导枯草芽孢生成,提取重组芽孢外衣蛋白进行电泳及免疫印迹分析,并使用NAP特异性抗体免疫荧光检测芽孢表面重组蛋白的表达.以普通芽孢作为对照组,使用表面表达NAP的重组枯草芽孢口服免疫小鼠,ELISA法检测小鼠免疫后0、15、30、45 d的NAP特异性血清IgG水平,判断重组芽孢免疫原性.结果 以pGEX-4T-1-NAP质粒为模板,扩增了NAP基因,并成功克隆入Pus 186-Cotc质粒,重组Pus 186-Cotc-NAP双酶切鉴定可见435 bp目的片段,测序结果显示NAP在正确读框中.营养耗竭法可诱导重组枯草芽孢生成,产量达1×10^11/L,提取芽孢外衣蛋白电泳见相对分子质量25 600的目的条带.免疫印迹显示使用NAP特异性抗体在相应相对分子质量25 600可见识别带.并且使用NAP特异性抗体可检测到重组芽孢免疫荧光,表明NAP在芽孢表面成功表达.与对照组相比,NAP重组芽孢口服免疫小鼠15d后,NAP特异性IgG升高即达高峰,30、45 d后一直维持在高峰平台期（均P＜0.05）,表明重组芽孢具免疫原性.结论 成功构建了表面表达NAP的枯草芽孢工程菌,重组芽孢口服免疫小鼠具免疫原性,为幽门螺杆菌枯草芽孢疫苗的研制提供了依据.

Objective To construct Bacillus subtilis spores which express Helicobacter pylori neutrophil activating protein （NAP） on surface and identify immunogenicity of recombinant spores.Methods NAP gene was amplified by specific primer by using constructed pGEX-4T-1-NAP plasmid as template.The PCR product was ligated with constructed Pus 186-Cotc plasmid,and transfected into Bacillus subtilis WB600.Recombinant plasmid was identified by double enzyme digestion and sequence analysis.Spores were made in Difco Sporulation Medium （DSM） by the exhaustion method.Coat protein of Bacillus subtilis was analyzed by SDS-PAGE and Western-blotting.Recombinant protein expression was identified by using NAP specific antibody in immunofluorescence assay.Mice were oral immunized by recombinant spores which expressed NAP on surface,and mice immunized with normal spores were used as control group.Immunogenicity of recombinant spores was identified by detecting NAP specific serum IgG by ELISA 0,15,30,45 days after immunization.Result NAP gene was successfully amplified from pGEX-4T-1-NAP plasmid and cloned with Pus186-Cotc plasmid.The 435 bp objective band was identified by double enzyme digestion.DNA sequence showed that NAP was in the correct open reading frame.Spores were made in DSM by the exhaustion method,about 1 × 10^11 spores were generated per litre of DSM culture medium.Recombinant fusion protein expression product could be seen at about 25 600.Recombinant fusion protein could be recognized by Western-blotting using NAP specific antibody.Immunofluorescence showed that NAP was expressed on spores surface.Oral administration of recombinant spores to mice induced NAP specific IgG response after 15 day of immunization.The response increased and the highest response was detected at 15 days after immunization.The response at 30 and 45 days time point stayed platform period compared with control group （all P〈0.05）,and this response showed the immunogenicity of recombinant spores.Conclusions Recombinant Bacillus subtilis spores whose surface expressing NAP are successfully constructed.Oral administration of recombinant spores in mice shows immunogenicity.Our study provides experimental evidence for Helicobacter pylori vaccine research using Bacillus subtilis spores.