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2025年4月3日 星期四
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嗜水气单胞菌外膜蛋白的原核表达及其对BALB/c小鼠的免疫保护研究
胡春霞1,李卫芬2,许梓荣3
1. 浙江大学饲料科学研究所;绍兴文理学院元培学院生命科学系
2. 浙江大学华家池校区动物科学学院
3.
Prokaryotic expression of outer membrane protein from Aeromonas hydrophila and the immunoprotection in BALB/c mice
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摘要 为确定嗜水气单胞菌(Aeromonas hydrophila,Ah)AS1.927株外膜蛋白OMP的表达产物是否具有抗原性及测定对小鼠的免疫保护力,根据GenBank上嗜水气单胞菌外膜蛋白ompA基因序列(登录号:AF146597)设计1对引物,克隆出ompA全长基因(1020 bp),经Sal I和Xhol I双酶切后连接pET-30a(+),转化大肠杆菌(Escherichia coli)BL21,IPTG诱导表达,经SDS-PAGE及Western blot分析鉴定。结果显示重组基因工程菌E.coli [pET-30a-ompA] 表达的融合蛋白可以被鼠抗嗜水气单胞菌外膜蛋白抗血清识别,说明重组蛋白OMP具有良好的免疫原性。将重组基因工程菌E.coli [pET-30a-ompA]口服免疫BALB/c小鼠;末次免疫1周后对小鼠肠粘膜SIgA水平(放射免疫分析)的检测结果揭示:该重组基因工程菌能提高SIgA的分泌(P<0.05);同时小鼠血清中测出特异性抗体(P<0.05)。末次免疫2周后用3.3 × 105 cfu的Ah AS1.927(100 LD50)腹腔注射攻击小鼠,重组蛋白对小鼠的相对免疫保护力(relative percent survival,RPS)为75%,表明重组基因工程菌可诱导小鼠产生一定的免疫保护作用。
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胡春霞
李卫芬
许梓荣
关键词 嗜水气单胞菌外膜蛋白原核表达小鼠免疫保护    
Abstract:To define whether the expressed ompA of Aeromonas hydrophila (Ah) AS1.927 strain has antigenicity and determine the relative percentage survival (RPS) for the mice, the 1020 bp fragment encoding ompA was cloned from the Ah AS1.927 genome with a pair of primers designed according to the sequence of omp in GenBank (AF146597)., After digesting the fragment with Sal I +Xhol I, the gene was inserted into pET-30a(+) vector. The recombinant pET-30a-ompA plasmid was transformed into the expressing strain BL21 and induced expression by IPTG. The recombinant protein ompA was analyzed by SDS-PAGE and Western blot, and results showed that the ompA fusion protein was about 40.7kD and could react with sera from mice infected with A. hydrophila. BALB/c mice were orally immunized at 1st, 2nd, 3rd, 20th, 21th, and 22th day with the engineering bacteria E.coli [pET-30a-ompA]. The serum IgG was detected by ELISA at 7days post-vaccination. On the 14th day after last immunization, the mice were challenged with 3.3 × 105 cfu of live Ah AS1.927 (100LD50). The results showed that mice vaccinated with E.coli [pET-30a-ompA] showed significantly increased antigen-specific IgG level in the serum (P<0.05) and SIgA level in the intestinal mucosa compared with the control and 75% relative percent survivals (RPS). This indicated that oral immunization of E.coli [pET-30a-ompA] could induce an immune response protective upon challenge with live Ah AS1.927 in BALB/c mice. The present study will provide technical support for developing of efficient gene engineering vaccine against A. hydrophila.
Key wordsAeromonas hydrophila    outer membrane protein    prokaryotic expression    mice    immune protection
收稿日期: 2009-02-26     
通讯作者: 李卫芬   
引用本文:   
胡春霞1,李卫芬2,许梓荣3. 嗜水气单胞菌外膜蛋白的原核表达及其对BALB/c小鼠的免疫保护研究[J]. , 2010, 18(2): 218-224.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2010/V18/I2/218
 
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