淡水小龙虾主要过敏原原肌球蛋白基因的克隆、表达及其免疫活性鉴定

Cloning and expression of Red swamp crayfish major allergen tropomyosin gene and identification of its immunoglogic activity

目的:克隆、表达淡水小龙虾肌肉中主要过敏原原肌球蛋白(tropomyosin),并对其免疫活性进行鉴定。方法:根据甲壳类的泛过敏原tropomyosin基因的高度保守性设计简并引物,通过RT-PCR克隆出淡水小龙虾虾肉中过敏原tropomyosin的全长基因。将该基因与pET-28a载体连接并转化大肠杆菌Rosetta(DE3),经诱导异丙基-B-D-硫代乳糖苷(IPTG)诱导表达,Ni2+亲和层析柱纯化后,用Western blot检测该重组蛋白的免疫活性。结果:序列分析显示该基因包括一个编码284个氨基酸的开放阅读框,与已知虾、蟹、龙虾等的过敏原tropomyosin基因有较高同源性(>80%)。根据过敏原的命名规则,将其命名为Proc 1,并提交GenBank数据库,登录号为FJ769183。重组小龙虾tropomyosin在大肠杆菌中能高效的表达,Western blot检测结果显示该重组蛋白具有良好的免疫活性。结论:研究成功表达了具有免疫活性的淡水小龙虾原肌球蛋白,为虾过敏性疾病的诊断和治疗奠定了基础。

Objective：To clone the major allergen tropomyosin gene from Red swamp crayfish and produce its recombinant protein and investigate its immunologic activity.Methods：Degenerate primers were designed according to the conserved sequence of tropomyosin.The RT-PCR was applied to clone the full-length tropomyosin allergen genes from Red swamp crayfish.The cDNA was sequenced and then subcloned into pET28a expression vector.The cloned tropomyosin cDNA gene was expressed in Escherichia coli Rosetta（DE3） by IPTG induction.The recombinant tropomyosin was purified by metal（Ni2＋） chelating affinity chromatography.The immunologic activity of the recombinant tropomyosin was examined by Western blot method.Results：Sequence analysis showed that this gene included an open reading frame coding for 284 amino acids and shared high identities（〉80%） with tropomyosin allergens from shrimp,crab and lobster.The deduced protein was therefore regarded as allergen and named as Pro c 1（EMBL/GenBank database entry No.FJ769183）.The recombinant allergen tropomyosin was highly expressed in Escherichia coli Rosetta（DE3）,Western blot analysis showed that the recombinant allergen has IgE-reactivity.Conclusion：The tropomyosin of Red swamp crayfish was successfully expressed and showed good immunologic activity,which laid a foundation of the specific diagnosis and immunotherapy in shrimp hypersensitivity disease.