口虾蛄原肌球蛋白基因表达及变应原性鉴定

Cloning,expression and purification of pan-allergen tropomyosin from Squilla oratoria and identification of its allergic activity

目的克隆口虾蛄原肌球蛋白(tropomyosin)基因并表达纯化出重组蛋白,研究其变应原性。方法提取口虾蛄总RNA,设计特异引物,通过反转录聚合酶链反应克隆出目的基因片段,测序后将该片段克隆到原核表达载体pET-28a上,转化到E.coliBL21(DE3)后,经异丙基-B-D-硫代乳糖苷(IPTG)诱导表达,用Ni2+亲和层析柱对重组变应原进行纯化。采用免疫印迹(Western-blot)检测其与对虾过敏的患者血清IgE结合活性。结果经序列测定,该基因含有长度为855bp的开放阅读框,编码284个氨基酸(GenBank登录号为EF584510)。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测该重组变应原在大肠埃希菌中高效表达36kDa的目的蛋白,且重组变应原具有良好的IgE结合活性。结论本研究成功获得了具有变应原活性的重组口虾蛄原肌球蛋白。

Objective To clone the tropomyosin gene from squilla oratoria and produce its recombinant protein and investigate its allergenicity. Methods The cDNA of tropomyosin was cloned using specific primers from the total RNA of squilla oratoria. The cloned gene was inserted into pMD18-T vector and digested by Nde I and HindIII. The cDNA was sequenced and then subcloned into pET-28a expression vector. The cloned tropomyosin cDNA gene was expressed in Escherichia coli BL21 （ DE3 ） by IPTG induction. The recombinant tropomyosin was purified by metal （ Ni^2 ＋ ） chelating affinity chromatography. The allergenicity of the recombinant tropomyosin was examined by western blotting method. Results The cloned cDNA ORF sequence contained 855 bp and encoded 284 amino acids （GenBank database entry No. EF584510）. The recombinant allergen tropomyosin was highly expressed in E. coli BL21 （ DE3 ） with the molecular weight of about 36 kDa under induction of IPTG and purified by 6-His-tag purification system. And the recombinant allergen was identified as its affinity to IgE antibodies from the shrimp-allergic patient sera. Conclusion Recombinant squilla oratoria tropomyosin with good allergenicity was obtained in this study.