重组红火蚁毒素致敏原Sol i1的表达及活性分析  

Expression and activity analysis of the recombinant Solenopsis invicta Buren venom allergen Sol i1

作　　者：韩雪清[1] 林祥梅[1] 张永国[2] 陈岩[1] 冶贵生[3] 杨伟东[4] 夏巧钰[1] 杨泽晓[3] 王建峰[1] 

机构地区：[1]中国检验检疫科学研究院,北京100029 [2]军事医学科学院微生物与流行病学研究所,北京100071 [3]西北农林科技大学动物科技学院,陕西杨凌712100 [4]深圳出入境检验检疫局,广东深圳518045

出　　处：《昆虫学报》2007年第7期655-661,共7页Acta Entomologica Sinica

基　　金：国家质检总局重点科研项目(2005IK169)

摘　　要：目的为研究红火蚁Solenopsis invicta Buren毒素蛋白致病分子机理,制备用于红火蚁蜇伤防治的制剂。方法本研究采用反转录PCR(RT-PCR)与套式PCR(nPCR)扩增出红火蚁体内毒素蛋白Sol i1全基因及其活性基因片段Sol i1a,进行测序与序列分析,构建重组质粒Sol i1-pET43.1a和Sol i1a-pET43.1a,经PCR、酶切和测序鉴定后转化BL21(DE3)进行IPTG诱导表达,对表达产物进行SDS-PAGE分析和Western blot检测后,用亲合层析法纯化,并将纯化的重组蛋白通过动物试验进行了致敏活性分析以及过敏体质治疗研究。结果本研究克隆的Sol i1基因序列与GenBank中红火蚁序列同源性为99 %,原核表达预期大小的2种重组蛋白能与组氨酸单抗发生特异性反应,且具有较高的致敏活性和良好的免疫治疗效果。结论原核表达的2种重组蛋白Sol i1和Sol i1a具有良好的生物活性,为红火蚁蜇伤的致病机理和防治研究奠定了基础。[Objective]To study the molecular pathogenic mechanism of Solenopsis invicta Buren venom allergens and produce reagents for the prophylaxis and therapy of S. invicta sting. [Methods] In the study, the S. invicta venom allergens sol i1 gene and its fragment sol i1a encoding the truncated peptide without transmembrane region were amplified by RT-PCR and nPCR,and then be sequenced. Two recombinant plasmids Sol i1-pET43.1a and Sol i1a-pET43.1a were constructed and verified by PCR, RE digestion and sequencing, and then transformed into E. coli BL21（DE3） and induced by IPTG. The expression products were purified by affinity chromatograph following with the identification of SDS-PAGE and Western blotting, and then used to inoculate rabbits for analyzing the allergenic activity and the potential in therapy. [Results] The results showed that the nucleotide homology of the amplified product with that published in GenBank was 99%, and the recombinant fusion proteins expressed in BL21（DE3） at high-level had good allergenic activity and immunotherapy potential. [Conclusions] Recombinant Sol i1 （rSol i1） and recombinant Sol i1a （rSol i1a） expressed in BL21（DE3） both had excellent biological activity, which set basis for further studying the mechanism of S. invicta venom allergens and the prevention of S. invicta.

关 键 词：红火蚁 基因 致敏原蛋白 表达 生物活性分析 

分 类 号：Q966[生物学—昆虫学]