多房棘球绦虫硫氧还蛋白过氧化物酶基因的克隆、表达及其免疫诊断价值初步评价

Cloning, expression, and determination of the immunodiagnostic value of thioredoxin peroxidase from Echinococcus multilocularis

目的克隆多房棘球绦虫(Echinococcus multilocularis,Em)硫氧还蛋白过氧化物酶(Thioredoxin peroxidase, TPx)基因,构建原核表达载体,诱导表达EmTPx蛋白,并初步评价其免疫诊断价值。方法从沙鼠中分离多房棘球绦虫原头蚴,提取总RNA,采用RT-PCR方法获取EmTPx的cDNA片段,克隆至原核表达载体pET-28a,构建重组表达质粒pET-28a-EmTPx,转化大肠埃希菌BL21进行目的蛋白的诱导表达及纯化,免疫小鼠制备抗EmTPx重组蛋白(rEmTPx)的多抗血清。通过Western blot对rEmTPx的免疫学特性进行鉴定,通过间接ELISA试验评价其免疫诊断价值。结果 PCR扩增EmTPx基因片段长度为582 bp,测序证实重组表达质粒pET-28a-EmTPx构建正确,表达的目的蛋白相对分子质量约为26×10^3,与理论值相符。Western blot显示该蛋白可被鼠抗rEmTPx多抗血清识别,以rEmTPx为抗原采用间接ELISA法检测AE患者血清,特异抗体的敏感性为66.2%,特异性为87.5%。结论成功构建了pET28a-EmTPx原核表达载体,表达蛋白具有Em原头蚴天然TPx的生物学功能表位,且具有一定的免疫学诊断价值,可作为AE血清学诊断的候选靶抗原。

Objectives To construct a prokaryotic expression vector, pET28 a-EmTPx, and to express a recombinant thioredoxin peroxidase protein, EmTPx, in order to evaluate its immunodiagnostic value. Methods Total RNA was extracted from Echinococcus multilocularis protoscoleces isolated from a Mongolian gerbil. cDNA was synthesized from the RNA, and the EmTPx gene was amplified with PCR using the cDNA as a template. The amplified DNA fragment was subcloned into an expression vector pET-28 a designated pET-28 a-EmTPx. The recombinant plasmid was transformed into E. coil BL21. The recombinant plasmid pet-28 a-emtpx was then sent to a company for sequencing. Expression of the recombinant expression plasmid pET-28 a-EmTPx was induced with IPTG, the expression products were analyzed using SDS-PAGE, and the recombinant protein EmTPx(rEmTPx) was purified on an Ni-NTA-His-Bind Resin affinity column. The purified rEmTPx was used as an antigen to immunize mice to produce anti-rEmTPx serum in order to test the biological activity of rEmTPx with Western blotting. Indirect ELISA was used to evaluate the immunodiagnostic value of rEmTPx. Results The EmTPx gene fragment was 582 bp in length. Sequencing verified that the recombinant expression plasmid pET-28 a-EmTPx was constructed correctly, and the expressed molecular mass of the target protein was about 26 ku, which was consistent with the theoretical value. Western blotting indicated that antiserum specifically recognized the rEmTPx protein and PSC antigen. ELISA results indicated that the sensitivity of rEmTPx was 66.22% and that its specificity was 87.50%. Conclusion The prokaryotic expression vector pET28 a-EmTPx was successfully constructed, and the expression products retained the biological functional epitope of native TPx of E. multilocularis. rEmTPx has a certain immunological diagnostic value and could serve as a target antigen for serological diagnosis of alveolar echinococcosis.