家蚕蚕蛹基因LOC101743840的原核表达、免疫学鉴定及生物信息学分析

Purification,immunological identification and bioinformatics study of gene:LOC101743840 from Bombyx mori pupae

目的原核表达家蚕蚕蛹LOC101743840(以下简称LOC)基因,鉴定LOC蛋白的免疫学特性,并进行生物信息学分析。方法将合成的家蚕蚕蛹LOC基因(gi|512917985)连接至克隆载体p MD18-T,用限制性内切酶Xho I和Bam H I分别对p MD18-T-LOC阳性质粒与原核表达载体p ET-28a双酶切,构建重组表达质粒p ET-28a-LOC并转化到E.coli BL21(DE3)中进行表达(经IPTG诱导);用Western blot鉴定重组LOC蛋白的免疫活性;用Clustalw2和MEGA5工具包分析LOC的基因;用Prot Param Tools预测LOC蛋白的理化性质;用PSIPRED和SWISS-MODEL预测其蛋白质结构。用IEDB、Preprod和DNAStar预测其细胞抗原表位。结果成功表达了家蚕蚕蛹LOC基因,其开放阅读框768 bp,编码255组氨基酸;由E.coli BL21(DE3)原核表达的重组LOC蛋白为可溶性,分子量约33 k D;Western bolt结果显示重组LOC蛋白可特异性结合家蚕蚕蛹过敏患者血清中的特异性Ig E;家蚕蚕蛹LOC与脐橙螟LOC106139919(gi|913330692)蛋白的同源性为69%。系统进化树显示家蚕蚕蛹与美国白蛾亲缘关系比较近。理化性质预测结果显示LOC蛋白质不稳定。蛋白质结构预测结果显示LOC主要的结构为无规则卷曲。T细胞抗原表位预测得到8个肽段(9-17、79-87、93-102、104-115、124-132、152-160、223-231和244-251)。B细胞抗原表位预测得到个5肽段(34-49、59-74、127-142、202-217和213-228)。结论成功表达了家蚕蚕蛹LOC,并证实重组LOC蛋白具有良好的免疫活性,为进一步研究家蚕蚕蛹蛋白的结构成分、免疫学特性及理化性质提供了实验数据。

Objective To purify,identify and analyze the protein of gene：LOC101743840 from Bombyx mori pupae.Methods The LOC gene（gi｜512917985） was synthesised and connected into p MD18-T.The p MD18-T-LOC was sub-cloned into PET-28 a vector by restriction endonuclease Eco R I and Xho I.Then,the recombinant plasmid Pet28a-LOC wastransformed into E.coli BL21（DE3）,which was induced with IPTG later.Western blot was used to test the immunogenicity ofLOC.Clustalw2 and MEGA5 were used to study the gene of LOC.Prot Param,PSIPRED and SWISS-MODEL were used topredict its protein structure.IEDB and Preprod were used to analyze the T cell antigen epitope.DNAStar was used to analyzethe B cell antigen epitope of LOC.Results After sequencing,the LOC gene was successfully cloned,and the open readingframe was 768 bp,encoding 255 amino acid group.After induction with IPTG,LOC protein was largely expressed and existedin soluble form,and the protein molecular weight was about 33 k Da.The Western blot results showed that LOC could bind withthe serum Ig E of B.mori allergic patients.Comparing the cloned B.mori LOC protein sequence with others,its homology with Amyelois transitella protein LOC106139919（gi｜913330692） was 69%.The molecular evolutionary tree analysis showed thatLOC had a close relationship with Hyphantria cunea.The physical and chemical properties prediction showed that LOC proteinwas unstable.The prediction of the secondary and tertiary structure indicated that LOC mainly consisted of random coil.Thebioinformatics analysis predicted eight T cell peptides（9-17,79-87,93-102,104-115,124-132,152-160,223-231,244-251） and five B cell peptides（34-49,59-74,127-142,202-217,213-228）.Conclusion LOC protein is successfullyexpressed in this study,and this study also confirms that the recombinant LOC protein has a good immunogenicity.The studyprovides a basis for further study of composition and physicochemical properties of B.mori allergen.