卫氏并殖吸虫TPx基因的克隆、表达和免疫学诊断价值

doi:10.12140/j.issn.1000-7423.2021.01.003

中国寄生虫学与寄生虫病杂志 ›› 2021, Vol. 39 ›› Issue (1): 20-26.doi: 10.12140/j.issn.1000-7423.2021.01.003

卫氏并殖吸虫TPx基因的克隆、表达和免疫学诊断价值

周岩¹(), 陈韶红¹, 李浩¹, 程娜¹, 洪加林², 许学年¹^,^*()

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

收稿日期:2020-07-27 修回日期:2020-10-10 出版日期:2021-02-28 发布日期:2021-03-10
通讯作者: 许学年
作者简介:周岩（1982-）女,硕士,助理研究员,从事寄生虫病防控研究。E-mail: zhouyan@nipd.chinacdc.cn
基金资助:
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)

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

ZHOU Yan¹(), CHEN Shao-hong¹, LI Hao¹, CHENG Na¹, HONG Jia-lin², XU Xue-nian¹^,^*()

1 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
2 People’s Hospital of Yongjia County, Yongjia 325100, China

Received:2020-07-27 Revised:2020-10-10 Online:2021-02-28 Published:2021-03-10
Contact: XU Xue-nian
Supported by:
Chinese Special Program for Scientific Research of Public Health(201502021);National Sharing Service Platform for Parasite Resources(20170804)

摘要/Abstract

摘要：

目的免疫筛选卫氏并殖吸虫成虫cDNA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λ ZAP cDNA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rPwTPx（全长型）和rPwTPx_1（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rPwTPx和rPwTPx_1,SDS-PAGE检测重组蛋白的表达情况。以rPwTPx、rPwTPx_1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rPwTPx和rPwTPx_1,相对分子质量（M_r）分别为25 000和22 000。以rPwTPx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rPwTPx_1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、0.033 ± 0.022、0.036 ± 0.015和0.032 ± 0.018。卫氏并殖吸虫病患者血清阳性率为88.9%（32/36）,健康人血清未检出阳性,15份华支睾吸虫病患者血清仅1份存在交叉反应,15份日本血吸虫病患者血清假阳性为3份,15份片形吸虫病患者和15份猪囊尾蚴病患者血清假阳性均为2份。重组蛋白rPwTPx的ELISA检测敏感性为58.3%,特异性为97.9%,总符合率为87.1%;rPwTPx_1则分别为88.9%、91.7%、90.9%。rPwTPx_1敏感性高于rPwTPx（P < 0.05）。以成虫粗抗原为检测抗原的ELISA检测结果显示,36份健康者血清A₄₅₀值为0.012,标准差为0.006。敏感性为100%,特异性为83.3%,总符合率为87.9%,与rPwTPx-ELISA和rPwTPx_1-ELISA检测的总符合率差异无统计学意义（P > 0.05）。结论筛选并克隆了卫氏并殖吸虫TPx基因,表达并纯化全长型和截短型两种重组TPx抗原,构建的全长型和截短型重组TPx抗原作为人体卫氏并殖吸虫病的免疫诊断抗原具有一定的诊断意义。

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

Abstract:

Objective 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 cDNA 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 (rPwTPx) and N-terminal truncated (rPwTPx_1) sequences of PwTPX were subcloned into the prokaryotic plasmid pET28a (+), respectively, to construct the expression vectors rPwTPX and rPwTPX_1. 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 rPwTPx and rPwTPx_1 were purified using the His-tagged affinity column (Ni-NTA). SDS-PAGE was used to analyzed the expression of the recombinant proteins. rPwTPx and rPwTPx_1 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 rPwTPx and rPwTPx_1 were obtained through expression and purification, with relative molecular mass M_r of 25 000 and 22 000, respectively. The rPwTPx 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, schistosomiasis japonica and cysticercosis patients. The cross-reaction with sera of fascioliasis gigantic patients was 2 samples. The ELISA using rPwTPx_1 as the antigen presented the A₄₅₀ values to the above testing sera was 0.144 ± 0.092, 0.022 ± 0.009, 0.027 ± 0.015, 0.033 ± 0.022, 0.036 ± 0.015 and 0.032 ± 0.018, respectively. The positive rate of paragonimiasis westermani patients sera was 88.9% (32/36), while the sera of healthy donors showed no cross-reaction. Only 1 serum sample of 15 clonorchiasis sinensis patients had cross-reaction. The cross-reaction with sera of schistosomiasis japonica, fascioliasis gigantic and cysticercariasis patients were 3, 2 and 2 samples. The total diagnostic coincidence rate, sensitivity and specificity of ELLSA using rPwTPx and rPwTPx_1 as antigen were 87.1%, 58.3%, 97.9%, and 90.9%, 88.9%, 91.7%, respectively. The sensitivity of the rPwTPx_1 ELISA was significantly higher than that of the rPwTPx ELISA (P < 0.05). Crude antigen-ELISA showed that the A₄₅₀ value of serum samples from the 36 healthy donors was 0.012, and S was 0.006. The sensitivity, specificity and total diagnostic coincidence rate of crude antigen were 100%, 83.3% and 87.9%, which did not significantly differ from those of rPwTPx and rPwTPx_1 (P > 0.05). Conclusion The gene encoding TPx was screened and expressed, and two structural types of recombinant protein were obtained, including full length and truncated types, which recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of P. westermani infection.

Key words: Paragonimus westermani, Thioredoxin peroxidase, Immunoscreen, Expression, ELISA

中图分类号:

R532.221

周岩, 陈韶红, 李浩, 程娜, 洪加林, 许学年. 卫氏并殖吸虫TPx基因的克隆、表达和免疫学诊断价值[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(1): 20-26.

ZHOU Yan, CHEN Shao-hong, LI Hao, CHENG Na, HONG Jia-lin, XU Xue-nian. Cloning, expression of the thioredoxin peroxidase gene of Paragonimus westermani and its immunodiagnostic potential[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2021, 39(1): 20-26.

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血清Serum	检测份数 No. detected	血清抗体阳性数No. of positive
血清Serum	检测份数 No. detected	rPwTPx	rPwTPx_1	成虫粗抗原 Crude antigen
卫氏并殖吸虫病患者Paragonimiasis westermani	36	21	32	36
华支睾吸虫病患者Clonorchiasis sinensis	15	0	1	5
日本血吸虫病患者Schistosomiasis japonica	15	0	3	1
片形吸虫病患者Fascioliasis gigantica	15	2	2	10
猪囊尾蚴病患者Cysticercosis	15	0	2	0
健康者 Healthy donors	36	0	0	0
合计Total	132	23	40	52

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

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