虾过敏原Troponin C (Pen m6)的克隆表达、免疫学鉴定及生物信息学分析

目的对斑节对虾(Penaeus monodon)第6组过敏原(Pen m6),肌钙蛋白C(Troponin C)进行克隆表达、免疫学鉴定并研究其生物学意义。方法提取斑节对虾总RNA;根据Gen Bank:HM034316.1设计引物及构建表达载体PET-24a-Pen m6;转化至大肠杆菌BL21(DE3)进行表达;纯化后的重组Pen m6用Western bolt来鉴定免疫学特性;用生物信息学相关工具对Pen m6进行同源性分析,并预测其蛋白质的结构和功能。结果克隆出斑节对虾Pen m6基因开放阅读框453 bp,编码150个氨基酸。重组Pen m6蛋白呈可溶性,分子量约35kDa,能够与斑节对虾过敏患者血清IgE结合。斑节对虾与凡纳滨对虾亲缘关系比较近,其中斑节对虾Pen m6蛋白与凡纳滨对虾Troponin C1(gb|AET36896.1|)同源性为98%。理化性质预测Pen m6蛋白质不稳定。蛋白质结构预测结果显示Pen m6的结构主要以α螺旋组成。T细胞抗原表位预测得到4个肽序列(15~23、35~43、91~99、112~120)。B细胞抗原表位预测得到4个肽序列(7~16、21~30、28~37、58~67)。结论克隆的重组斑节对虾Pen m6蛋白具有良好的免疫原性,为进一步研究斑节对虾过敏原的结构成分及其理化性质奠定理论基础。

Clone, expression, purification, immunological identification and bioinformatics analysis of allergen Troponin C (Pen m6) in Penaeus monodon

Objective To clone and express the Troponin C (Pen m6) gene, of Penaeus monodon, purify and identify its immunogenicity and investigate its biological significances. Methods Total RNA was extracted from standardized cultivated Penaeus monodon. The primers, designed according to sequences (GenBank: HM034316.1) were amplified by RT-PCR. The PCR product was sub cloned into PET-24a vector. The PET24a- Pen m6 was transformed into E. coli BL21(DE3). Western bolt technique was used to test the immunogenicity of Pen m6. Bioinformatics tools were used to perform homology analysis and predict the structure and function of Pen m6. Results Totally 453 bp open reading frame of the Pen m6 gene was cloned, and 150 amino acids were encoded. After inducted with IPTG, Pen m6 protein which existed in soluble form was largely expressed, and the protein molecular weight was about 35 kDa. The Western bolt results showed that Pen m6 could bind with Penaeus monodon allergic patients serum IgE. Comparing the cloned Penaeus monodon Pen m6 protein sequence with others, we found that its homology with Litopenaeus vannamei Troponin C1 (gb| AET36896.1|) was 98%. The molecular evolutionary tree analysis showed that Penaeus monodon had a close relationship with Litopenaeus vannamei. The physical and chemical properties prediction showed Pen m6 protein was unstable. The prediction of the secondary and tertiary structure indicated that Pen m6 mainly consisted of helix. Bioinformatics analysis predicted 4 peptides (15~23, 35~43, 91~99, 112~120) as the T cell epitopes and 4 peptides (7~16, 21~30, 28~37, 58~67) as the B cell epitopes. Conclusion The recombinant Pen m6 protein has good immunogenicity. The study provides a basis for further study of composition and physicochemical properties of Penaeus monodon allergen.