家蚕蚕蛹过敏原表皮蛋白RR-2基序63前体(CPR63)的克隆表达、纯化鉴定及生物信息学分析

Clone,expression,purification,immunological identification and bioinformatics analysis of allergen cuticular protein RR-2 motif 63 precursor(CPR63) of silkworm(Bombyx mori) pupae

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。

Bombyx mori motif 63 precursor（CPR63） gene for immunogenicity and bioinformatics analysis of the CPR63 protein.The gene ofBombyx mori pupae CPR63（ref｜NM_001173223.1｜） was synthesized and connected into p MD18-T to constructp MD18-T-CPR63 plasmid,which was then subcloned into PET-44 a vector by restriction endonuclease EcoR Ⅰand Xho Ⅰ.The recombinant plasmid p ET44a-CPR63 was transformed into E.coli BL21（DE3） and induced withIPTG for expression.Western bolt technique was used to test the immunogenicity of CPR63 and the clustalw2 wasused to conducte homology analysis while MEGA5 was applied to construct phylogenetic tree of CPR63;Prot Paramtools were used to predict the physical and chemical properties of CPR63;the protein structure of CPR63 waspredicted by PSIPRED and SWISS-MODEL;IEDB and Preprod were used to analyze the T cell antigen epitope,while DNAStar was used to analyze the B cell antigen epitope of CPR63.Sequencing analysis confirmed that theCPR63 gene has successfully cloned,and the open reading frame is 549 bp,encoding 182 amino acid groups.Afterinduction with IPTG,CPR63 protein was largely expressed by E.coli BL21（DE3） in soluble form,with proteinmolecular weight of about 20 k Da.The Western boltresults showed that CPR63 can bind with Bombyx mori allergic patient serum Ig E.The cloned Bombyx mori CPR63 protein sequence demonstrated a homology of85% with that of Danaus plexippus cuticular proteinRR-2 motif 63（gb｜EHJ69818.1｜）.The molecular evolutionary tree analysis showed that CPR63 had a closerelationship with Plutella xylostella.The physical and chemical properties prediction showed CPR63 protein wasstable.The prediction of the secondary and tertiary structure indicated that CPR63 mainly consisted of random coil.Bioinformatics analysis predicted four peptides（5-13,20-28,51-59,87-95） as the T cell epitopes and fourpeptides（21-40,37-56,47-66,64-83） as the B cell epitopes.In conclusion,we successful cloned and expressedCPR63,and confirmed that the recombinant CPR63 protein has good immunogenicity.The study provides a basis forfurther study of composition and physicochemical properties of Bombyx mori allergen.