卫氏并殖吸虫TPx基因的克隆、表达和免疫学诊断价值  

Cloning,expression of the thioredoxin peroxidase gene of Paragonimus westermani and its immunodiagnostic potential

作　　者：周岩 陈韶红 李浩 程娜 洪加林 许学年 ZHOU Yan;CHEN Shao-hong;LI Hao;CHENG Na;HONG Jia-lin;XU Xue-nian(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Chinese Center for Tropical Diseases Research,WHO Collaborating Center for Tropical Diseases,National Center for International Research on Tropical Diseases,Ministry of Science and Technology,Key Laboratory of Parasite and Vector Biology,Ministry of Health,Shanghai 200025,China;People's Hospital of Yongjia County,Yongjia 325100,China)

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,国家热带病研究中心,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海200025 [2]永嘉县人民医院,永嘉325100

出　　处：《中国寄生虫学与寄生虫病杂志》2021年第1期20-26,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家公益性卫生行业科研专项(201502021);国家寄生虫种质资源共享服务平台(20170804)。

摘　　要：目的免疫筛选卫氏并殖吸虫成虫c DNA表达文库,克隆并表达卫氏并殖吸虫硫氧还蛋白过氧化物酶(TPx),初步评价其免疫诊断价值。方法用卫氏并殖吸虫病患者的混合血清筛选卫氏并殖吸虫成虫λZAP c DNA表达文库,取阳性噬菌体进行克隆、测序和序列比对分析。将TPx基因全长片段和前端截短片段分别克隆至原核表达质粒pET28a (+)中,构建r Pw TPx (全长型)和r Pw TPx1 (截短型)表达载体。构建的重组蛋白经1 mmol/L的异丙基-β-D-半乳糖苷(IPTG)诱导表达。使用蛋白抽提剂、溶菌酶和核酸酶裂解表达细菌,用组氨酸标签亲和纯化柱(Ni-NTA树脂)纯化r Pw TPx和r Pw TPx1, SDS-PAGE检测重组蛋白的表达情况。以r Pw TPx、r Pw TPx1和粗抗原作为检测抗原,间接ELISA法检测36份卫氏并殖吸虫病、 15份华支睾吸虫病、 15份日本血吸虫病、 15份片形吸虫病和15份猪囊尾蚴病的患者血清,以及36份健康人血清,评价该重组蛋白的免疫诊断价值。以健康人血清对照组的平均值加上4个标准差作为阳性判断值。所有数据均采用SAS 9.2软件进行统计学分析。结果克隆的卫氏并殖吸虫TPx基因,经原核表达、纯化,获得其可溶性重组蛋白r Pw TPx和r Pw TPx1,相对分子质量(Mr)分别为25 000和22 000。以r Pw TPx作为检测抗原的ELISA检测结果显示,卫氏并殖吸虫病患者、健康者、华支睾吸虫病患者、日本血吸虫病患者、片形吸虫病患者和猪囊尾蚴病患者血清组的吸光度(A450值)分别为0.150±0.092、 0.036±0.014、 0.043±0.019、 0.047±0.013、 0.060±0.022和0.048±0.021。卫氏并殖吸虫病患者血清阳性率为58.3%(21/36), 36份健康人血清、 15份华支睾吸虫病患者血清、 15份日本血吸虫病患者血清、 15份猪囊尾蚴病患者血清均未检出阳性,15份片形吸虫病患者血清假阳性为2/15。以r Pw TPx1作为检测抗原ELISA检测结果则为0.144±0.092、 0.022±0.009、 0.027±0.015、 0Objective To immunologically screen the cDNA library of adult Paragonimus westermani for the gene encoding thioredoxin peroxidase(TPx), clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. Methods The λ ZAP c DNA library was immunologically screened with the pooled serum of P. westermani patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment.The full length(r Pw TPx) and N-terminal truncated(r Pw TPx1) sequences of Pw TPX were subcloned into the prokaryotic plasmid pET28 a(+), respectively, to construct the expression vectors r Pw TPX and r Pw TPX1. The expression of the two constructed recombinant plasmids was induced by 1 mmol/L IPTG. Protein extraction reagents,lysozyme and nuclease were used to lyse and express the bacterial fluid. The recombinant proteins of r Pw TPx and r Pw TPx1 were purified using the His-tagged affinity column(Ni-NTA). SDS-PAGE was used to analyzed the expression of the recombinant proteins. r Pw TPx and r Pw TPx1 were used to test the sera of 36 paragonimiasis westermani patients, 15 patients with clonorchiasis sinensis, 15 schistosomiasis japonica patients, 15 fascioliasis gigantica patients, 15 cysticercosis patients, and 36 healthy donors, using the indirect ELISA method. Data were analyzed with the SAS 9.2 software. Results The TPx recombinant proteins r Pw TPx and r Pw TPx1 were obtained through expression and purification, with relative molecular mass Mrof 25 000 and 22 000, respectively. The r Pw TPx ELISA results showed that the A450 values for the sera from patients of paragonimiasis westermani, healthy persons,clonorchiasis sinensis, schistosomiasis japonica, fascioliasis gigantic, and cysticercosis patients were 0.150 ± 0.092,0.036 ± 0.014, 0.043 ± 0.019, 0.047 ± 0.013, 0.060 ± 0.022 and 0.048 ± 0.021, respectively, The positive rate in serum of paragonimiasis westermani patients was 58.3%(21/36). There was no cross-reaction with sera of healthy donors, clonorchiasis sinensis, schisto

关 键 词：卫氏并殖吸虫 硫氧还蛋白过氧化物酶 免疫学筛选 原核表达 ELISA 

分 类 号：R532.221[医药卫生—内科学]