家蚕过敏原细胞视黄酸结合蛋白(CRABP)的克隆表达及生物学分析

Cloning,Expression and Bioinformatics Analysis of Cellular Retinoic Acid Binding Protein(CRABP)in Silkworm,Bombyx Mori

目的研究家蚕细胞视黄酸结合蛋白(CRABP)的原核克隆表达及生物学意义。方法家蚕总RNA提取;根据ref|NM-001043899|设计引物;RT-PCR扩增;PET-28a质粒目的基因表达载体的构建,转化大肠杆菌BL21(DE3);Western bolt检测重组CRABP;采用MEGA5工具包和clustalw2进行同源性分析;采用ProtParam Tools预测CRABP的理化性质;采用PSIPRED和SWISS-MODEL预测其蛋白质结构。利用Preprod、IEDB和DNAStar对CRABP蛋白T、B细胞抗原表位进行预测。结果克隆出家蚕CRABP基因开放阅读框399bp,编码132个氨基酸。CRABP工程菌(DE3)经IPTG诱导高效表达CRABP重组的可溶性蛋白,分子量约18kDa。重组CRABP能够与家蚕过敏患者血清IgE结合。测序证明克隆的家蚕CRABP蛋白与黑脉金斑蝶细胞视黄酸结合蛋白(gb|EHJ79039.1|)同源性为94%。系统进化树结果显示家蚕与小菜蛾亲缘关系比较近。理化性质预测显示CRABP蛋白质较稳定。二级结构及三级结构预测结果显示CRABP的结构主要以β折叠组成。T细胞抗原表位预测得到4个肽序列(4-12、20-29、51-59、80-88)。B细胞抗原表位预测得到4个肽序列(1-16、40-55、67-82、105-120)。结论克隆并表达的家蚕CRABP蛋白具有良好的变应原性,为进一步研究家蚕过敏原的结构成分及其理化性质奠定理论基础。

Objective To study the cloning, expression and biological significance of cellular retinoic acid binding protein（CRABP） in Bombyx mori. Methods Total RNA was extracted from Bombyx mori. The cDNA was amplified by using RT-PCR with primers designed according to sequences（tel｜ NM-001043899｜）. The PCR product was subcloned into PET-28a vector and the PET28a-CRABP was transformed into E. coli BL21. The immunogenicity of CRABP was tested by Western bolt. The homology was analyzed by MEGA5 and clustalw2. The physical and chemical properties were predicted by ProtParam tools. The protein structure was predicted by PSIPRED and SWISS-MODEL. T and B cell epitopes were predicted by Preprod, IEDB and DNAStar. Results The cloned CRABP gene contained an open reading flame of 399 bp which encoded a pro-tein of 132 amino acids. After induction by IPTG,soluble CRABP protein was highly expressed in engineered CRABP bacteria（DE3） with a molecular weight of 18 kDa. The recombinant CRABP could bind serum IgE in silkworm-allergic patients. Sequencing confirmed that the cloned Bombyx mori CRABP protein shared 94% homology with Danaus plexippus CRABP（gb｜EHJ79039.1）. The molecular evolutionary tree analysis showed that CRABP had a close relationship with Plutella xylostella. The physicochemical property prediction showed that CRABP protein was stable. The secondary and tertiary structure prediction indicated that CRABP mainly consisted of 13-sheets. Bioinformatics analysis predicted four peptides（4-12,20-29,51-59,80-88） as the T cell epitopes and four peptides（1-16,40-55,67-82,105-120） as the B cell epitopes. Conclusion The cloned and expressed CRABP protein has good immunogenicity. Our results provide a theoretical basis for further study of the composition and physicochemical property of Bombyx mori allergen.