论文部分内容阅读
目的克隆表达家蚕蚕蛹表皮蛋白RR-2基序63前体(CPR63)基因,纯化鉴定蛋白的过敏原性,并进行生物信息学分析。方法合成家蚕蚕蛹CPR63蛋白的基因(ref|NM_001173223.1|),将其连接至克隆载体p MD18-T,p MD18-T-CPR63阳性质粒与原核表达载体p ET-44a分别用限制性内切酶酶切,构建出重组表达质粒p ET-44a-CPR63,转化到大肠杆菌BL21(DE3)中经IPTG诱导表达;Western blot检测重组CPR63蛋白特异性过敏原性;用clustalw2和MEGA5工具包进行同源性分析;用Prot Param Tools预测其理化性质;用PSIPRED和SWISS-MODEL预测其蛋白质结构。利用网络服务器IEDB、Preprod和DNAStar,对CPR63蛋白T、B细胞抗原表位进行预测。结果经测序鉴定,本研究成功克隆出了家蚕蚕蛹CPR63基因,其开放阅读框549 bp,编码182组氨基酸。通过大肠杆菌E.coli BL21(DE3)表达CPR63重组蛋白主要以可溶性形式存在,相对分子质量约20 000。Western blot显示重组CPR63能够与家蚕蚕蛹过敏患者血清Ig E结合。家蚕蚕蛹CPR63蛋白与黑脉金斑蝶表皮蛋白RR-2基序63(gb|EHJ69818.1|)同源性为85%。系统进化树结果显示家蚕蚕蛹与小菜蛾亲缘关系比较近。理化性质预测显示CPR63蛋白质较稳定。二级结构及三级结构预测结果显示CPR63的结构主要以无规则卷曲组成。T细胞抗原表位预测得到4个肽序列(5~13、20~28、51~59和87~95)。B细胞抗原表位预测得到4个肽序列(21~40、37~56、47~66和64~83)。结论成功克隆并表达了家蚕蚕蛹CPR63,并证实重组CPR63蛋白具有良好的免疫原性,为进一步研究家蚕蚕蛹过敏原的结构成分及其理化性质奠定理论基础。
Objective To clone the CPR63 gene of silkworm silkworm pupal epidermal protein RR-2 and purify its allergenicity by bioinformatics analysis. Methods The CPR63 protein of silkworm pupa (ref | NM_001173223.1) was synthesized and ligated into the cloned vector p MD18-T, p MD18-T-CPR63 positive plasmid and the prokaryotic expression vector p ET-44a respectively by restriction endonuclease The recombinant plasmid p ET-44a-CPR63 was constructed and transformed into E. coli BL21 (DE3) and induced by IPTG. Western blot was used to detect the specific allergenicity of recombinant CPR63 protein. The clustalw2 and MEGA5 kit Source analysis; Prot Param Tools prediction of its physico-chemical properties; PSIPRED and SWISS-MODEL prediction of its protein structure. The CPR63 protein T and B cell epitopes were predicted by web server IEDB, Preprod and DNAStar. Results After sequencing, the CPR63 gene of silkworm silkworm chrysalis was cloned successfully. The open reading frame of the CPR63 gene was 549 bp, encoding 182 amino acids. The recombinant protein CPR63 expressed in E. coli BL21 (DE3) mainly exists in soluble form with the molecular weight of about 20,000. Western blot showed that recombinant CPR63 could bind IgE in serum of silkworm pupae allergy patients. The homology of silkworm chrysalis pupae CPR63 protein with monarch epidermal protein RR-2 motif 63 (gb | EHJ69818.1 |) was 85%. The results of phylogenetic tree showed that silkworm pupae had relatively close relationship with diamondback moth. Physical and chemical properties predicted CPR63 protein is more stable. Secondary structure and tertiary structure prediction results show that the structure of CPR63 is mainly composed of random curl. Four peptide sequences (5-13, 20-28, 51-59 and 87-95) were predicted for T cell epitopes. Four peptide sequences were predicted from B cell epitopes (21-40, 37-56, 47-66 and 64-83). Conclusion CPR63 was successfully cloned and expressed in silkworm silkworm chrysalis, and confirmed that recombinant CPR63 protein has good immunogenicity, which laid a theoretical foundation for further study on the structural components and physicochemical properties of silkworm pupae allergens.