腐食酪螨Tyr p 34基因的克隆表达、纯化及免疫原性鉴定  

Clonal expression, purification and immunogenicity identification of the Tyrophagus putrescentiae Tyr p 34 gene

作　　者：王昱嘉 陈德盛 袁如意 钟永浩 刘晓宇[1] WANG Yujia;CHEN Desheng;YUAN Ruyi;ZHONG Yonghao;LIU Xiaoyu(Institute for Allergy and Immunology,Shenzhen University,Guan gdong,Shenzhen 518060,China)

机构地区：[1]深圳大学过敏反应与免疫学研究所,广东深圳518060

出　　处：《中国医药科学》2022年第13期22-26,共5页China Medicine And Pharmacy

基　　金：国家自然科学基金(31729002)。

摘　　要：目的 克隆腐食酪螨Tyr p 34基因,表达、纯化出重组蛋白,并鉴定其免疫原性。方法 提取腐食酪螨的总RNA,通过反转录聚合酶链式反应获得其cDNA片段。根据引物和cDNA进行PCR扩增,通过酶切连接构建表达载体pET-28a(+)-Tyr p 34,并将鉴定为阳性的重组表达质粒转入感受态大肠杆菌中。分别在高温及低温条件下,少量诱导表达重组蛋白Tyr p 34以进行蛋白鉴定;大量诱导表达该重组蛋白并采用亲和层析法纯化,将纯化后的目的蛋白用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测并进行免疫印迹分析(Western blot)及生物信息学分析。结果 双酶切结果显示Tyr p 34基因与载体成功连接。少量表达后进行蛋白鉴定,显示目的基因在大肠杆菌中成功表达,Tyr p 34蛋白主要以可溶性蛋白的形式存在,在高温组中含量相对较多。大量表达后纯化的重组Tyr p 34蛋白分子量约为20 kDa。Western blot显示有明显条带,表明该重组基因所表达的蛋白可与腐食酪螨过敏患者的血清反应。生物信息学分析结果显示Tyr p 34基因大小为462 bp,编码153个氨基酸。氨基酸序列与NCBI公布的Tyr p 34氨基酸序列(登录号:ACL36923.1)同源性为99%。Tyr p 34蛋白有5个磷酸化位点,主要由无规则卷曲和α-螺旋构成。结论腐食酪螨Tyr p 34基因成功克隆并表达、纯化出重组蛋白,该蛋白具有免疫原性。通过Tyr p 34的生物信息学分析,有利于深入研究腐食酪螨过敏原的结构和理化性质。Objective To clone the Tyr p 34 gene of Tyrophagus putrescentiae, express and purify the recombinant protein, and identify its immunogenicity. Methods Total RNA of Tyrophagus putrescentiae was extracted and its complementary DNA(cDNA) fragments were obtained by reverse transcription-polymerase chain reaction(PCR). PCR amplification was performed according to the primers and cDNA, and the expression vector pET-28a(+)-Tyr p 34 was constructed by enzymatic ligation, and the identified positive recombinant expression plasmids were transferred into competent Escherichia coli(E. coli). Under high and low temperature,a small amount of the recombinant Tyr p 34 protein was induced to express for protein identification, and a large amount of the recombinant Tyr p 34 protein was induced to express and purified by affinity chromatography. The purified target proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and were subjected to Western blot analysis and bioinformatics analysis. Results The double digestion results showed that the Tyr p 34 gene was successfully ligated to the vector. Protein identification after a small amount of expression showed that the target gene was successfully expressed in E. coli and the Tyr p 34 protein was mainly in the form of soluble protein, which was relatively abundant in the high temperature group. The recombinant Tyr p 34 protein purified after a large amount of expression had a molecular weight of about 20 kDa. Obvious bands were found by Western blot, indicating that the protein expressed by this recombinant gene could react with the serum of patients with Tyrophagus putrescentiae allergy. Bioinformatics analysis showed that the Tyr p 34gene was 462 bp in size and encodes 153 amino acids, and its amino acids sequence was 99% homologous to the National Center for Biotechnology Information(NCBI) published Tyr p 34 gene sequence(accession number:ACL36923.1). The Tyr p 34 protein had five phosphorylation sites and consisted mainly of random coils and

关 键 词：腐食酪螨 Tyr p 34 蛋白鉴定 免疫原性 生物信息学分析 

分 类 号：R392[医药卫生—免疫学]