细粒棘球绦虫钙网蛋白的原核表达及功能初步鉴定

doi:10.12140/j.issn.1000-7423.2025.02.012

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (2): 223-230.doi: 10.12140/j.issn.1000-7423.2025.02.012

细粒棘球绦虫钙网蛋白的原核表达及功能初步鉴定

普娜¹^,²(), 薄新文¹^,², 赵文卿¹, 陈旭珂¹^,², 张玉霞¹^,², 张艳艳¹, 孙艳¹, 王正荣¹^,^*()()

1 省部共建绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院畜牧兽医研究所，新疆石河子 832000
2 石河子大学动物科技学院，新疆石河子 832000

收稿日期:2024-10-28 修回日期:2025-01-15 出版日期:2025-04-30 发布日期:2025-02-25
通讯作者: * 王正荣（ORCID：0000-0001-7169-2567），男，硕士，副研究员，从事寄生虫学与寄生虫病研究，E-mail：wzrtiger@sina.com
作者简介:普娜，女，硕士研究生，从事寄生虫学与寄生虫病研究。E-mail：3437324286@qq.com
基金资助:
国家自然科学基金(32360887);国家自然科学基金(32460894);新疆生产建设兵团国际科技合作(2021BC008);省部共建绵羊遗传改良与健康养殖国家重点实验室重大专项(2021ZD02);兵团农业科技创新工程专项(NCG202213)

Prokaryotic expression and preliminary functional identification of Echinococcus granulosus calreticulin

PU Na¹^,²(), BO Xinwen¹^,², ZHAO Wenqing¹, CHEN Xuke¹^,², ZHANG Yuxia¹^,², ZHANG Yanyan¹, SUN Yan¹, WANG Zhengrong¹^,^*()()

1 The State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding/Institute of Animal Husbandry and Veterinary Medicine, Xinjiang Academy of Agricultural Reclamation Sciences, Shihezi 832000, Xinjiang, China
2 College of Animal Science and Technology of Shihezi University, Shihezi 832000, Xinjiang, China

Received:2024-10-28 Revised:2025-01-15 Online:2025-04-30 Published:2025-02-25
Contact: * E-mail：wzrtiger@sina.com
Supported by:
National Natural Science Foundation of China(32360887);National Natural Science Foundation of China(32460894);international scientific and technological cooperation of Xinjiang Production and Construction Corps(2021BC008);major project of the State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding(2021ZD02);Agricultural Science and Technology Innovation Project of the Corps(NCG202213)

摘要/Abstract

摘要：

目的原核表达细粒棘球绦虫钙网蛋白，并对其功能进行初步探索。方法以细粒棘球绦虫原头节cDNA为模板，PCR扩增目的基因，连接至pET-28a（+）载体，构建重组质粒pET28a-CRT，将测序正确的重组质粒转化至表达菌BL21诱导表达，经十二烷基硫酸钠聚丙烯酰胺（SDS-PAGE）电泳以及蛋白质免疫印迹（Western blotting）分析鉴定rEgCRT；应用在线生物信息学分析软件分析EgCRT表达蛋白理化性质及部分生物学功能；rEgCRT免疫小鼠制备多克隆抗体，并用间接ELISA和Western blotting检测其抗体效价及反应原性；应用间接免疫荧光分析CRT在原头节和成虫体内的分布以及rEgCRT与犬小肠上皮细胞的结合；应用RT-qPCR方法分析crt基因在虫体不同发育阶段转录水平及rEgCRT（实验组，PBS为对照组）对犬小肠上皮细胞凋亡、焦亡的影响。结果 CRT蛋白分子式为C₂₀₃₃H₃₀₅₁N₅₁₁O₆₅₀S₉，预测EgCRT蛋白α-螺旋占24.05%、无规则卷曲占60.00%，延伸链结构占15.95%。EgCRT与多房棘球绦虫和猪带绦虫的CRT在系统进化树上聚在一大分支。成功诱导表达纯化获得纯度较高且相对分子量约为50 000的rEgCRT；免疫学特性鉴定表明rEgCRT具有较好的抗原特异性；间接免疫荧光试验发现EgCRT可分别定位于原头节表皮层和成虫表皮层及部分实质组织。RT-qPCR分析结果显示，成虫阶段EgCRT的表达水平（24.83 ± 0）高于原头节阶段（27.05 ± 0.008）（t = 45.76，P < 0.01）。间接免疫荧光分析结果显示，rEgCRT共孵育的犬小肠上皮细胞有绿色荧光聚集。RT-qPCR结果显示，培养24 h后实验组和对照组Bax相对转录水平分别为31.63 ± 0.001、24.17 ± 0.027（t = 7.94，P < 0.01），表达上调；Bcl-2相对转录水平分别为34.55 ± 0.070、19.65 ± 0.042（t = 15.70，P < 0.01），表达下调；IL-18相对转录水平分别为25.62 ± 0.067、20.93 ± 0.014（t = 5.05，P < 0.01），表达下调；IL-1β相对转录水平分别为28.18 ± 0.001、28.71 ± 0.020（t = 0.26，P > 0.05），表达下调。结论 rEgCRT具有良好的抗原性，EgCRT在细粒棘球绦虫不同虫期均有表达，且其可能在细粒棘球绦虫固着、定植或生长发育中具有重要的作用；此外，rEgCRT与犬SIECs发生黏附并调节细胞的凋亡、焦亡，可能参与感染过程或宿主免疫应答，具有作为潜在疫苗候选分子、药物靶标的应用前景。

关键词: 细粒棘球绦虫, 钙网蛋白, 克隆表达, 免疫荧光, 分子功能

Abstract:

Objective To express Echinococcus granulosus (Eg) calreticulin (CRT) in prokaryotic cells and preliminarily explore the function of EgCRT. Methods The cDNA of Eg protoscoleces was used as a template to amplify the target gene using PCR assay, which was ligated into the pET-28a (+) vector to construct the recombinant plasmid pET28a-CRT. The recombinant plasmid was transformed into the Escherichia coli BL21 strain. The rEgCRT was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and the physicochemical properties and some biological functions of the EgCRT protein were predicted using online bioinformatics tools. Mice were immunized with rEgCRT to prepare the polyclonal antibody, and the antibody titer and reactogenicity were detected using indirect ELISA and Western blotting, respectively. The distribution of CRT in protoscoleces and adults and the binding of rEgCRT to canine intestinal epithelial cells were analyzed using indirect immunofluorescence. In addition, the relative transcription level of the crt gene at different developmental stages and the effect of rEgCRT (experimental group and PBS as a control group) on apoptosis and pyroptosis of canine intestinal epithelial cells were examined using real-time quantitative reverse transcription PCR (RT-qPCR) assay. Results The molecular formula of the CRT protein was C₂₀₃₃H₃₀₅₁N₅₁₁O₆₅₀S₉, and the EgCRT protein was predicted to contain α-helix (24.05%), irregular coils (60.00%) and extended chain structures (15.95%). EgCRT, along with the CRTs of E. multilocularis and Taenia solium, were clustered into a clade on the phylogenetic tree. The rEgCRT protein was successfully expressed and highly purified, with a relative molecular weight of approximately 50 000. The rEgCRT protein showed a high antigenic specificity, and indirect immunofluorescence assay showed that EgCRT was localized in the epidermis of protoscoleces and adults and some parenchyma tissues. RT-qPCR assay showed higher expression of EgCRT in the adult stage (24.83 ± 0) than in the protoscoleces stage (27.05 ± 0.008) (t = 45.76, P < 0.01), and indirect immunofluorescence assay showed that the canine small intestinal epithelial cells co-cultured with rEgCRT presented green fluorescence aggregation. RT-qPCR assay showed that the relative transcription levels of the Bax gene were (31.63 ± 0.001) and (24.17 ± 0.027) in the experimental group and the control group 24 hours post-culture (t = 7.94, P < 0.01), indicating up-regulated expression, and the relative transcription levels of Bcl-2 were (34.55 ± 0.070) and (19.65 ± 0.042) (t = 15.70, P < 0.01), indicating downregulated expression, while the relative transcription levels of IL-18 were (25.62 ± 0.067) and (20.93 ± 0.014) (t = 5.05, P < 0.01), indicating downregulated expression, and the relative transcription levels of IL-1β were (28.18 ± 0.001) and (28.71 ± 0.020) (t = 0.26, P > 0.05), indicating downregulated expression. Conclusion The rEgCRT protein has a high antigenicity, and EgCRT is expressed in different developmental stages of E. granulosus, and may play an important role in the fixation, colonization, growth or development of E. granulosus. In addition, rEgCRT adheres to canine small intestinal epithelial cells and regulates cell apoptosis and pyroptosis, which may be involved in the process of E. granulosus infections or host immune responses, and has a promise as potential vaccine candidates and drug targets.

Key words: Echinococcus granulosus, Calreticulin, Cloning expression, Immunofluorescence, Molecular function

中图分类号:

R383.3

普娜, 薄新文, 赵文卿, 陈旭珂, 张玉霞, 张艳艳, 孙艳, 王正荣. 细粒棘球绦虫钙网蛋白的原核表达及功能初步鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(2): 223-230.

PU Na, BO Xinwen, ZHAO Wenqing, CHEN Xuke, ZHANG Yuxia, ZHANG Yanyan, SUN Yan, WANG Zhengrong. Prokaryotic expression and preliminary functional identification of Echinococcus granulosus calreticulin[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(2): 223-230.

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基因Gene	引物序列（5'→3'） Primer sequences (5'→3')	片段大小/bp Product length/bp
EgCRT	F：CCG GAATTCTACGACTATAATAATGATGC-CGAAC	1 108
	R：CCG CTCGAGCTTCTCTCCCAAAAGGGTCAT
qCRT	F：TCGCATTGTTATTCGTTGTT	150
	R：GGCATCTTGTGTTGTTCGT
GAPDH^a	F：CGCATCGGTCGTCTTGTGTT	190
	R：CGGTAATCCTGTGGCTGTCAAT
Bax	F：GATGAACTGGACAGTAACATGGA	158
	R：AGTTTGCTGGCAAAGTAGAAGAG
Bcl-2	F：GATTGTGGCCTTCTTTGAGTTC	168
	R：TACAGTTCCACAAAGGCATCC
IL-1β	F：TTGTAGCTTTGGAGAAGCTGAAG	179
	R：GTGGCTTATGTCCTGTAACTTGC
IL-18	F：AGGATATGCCCGATTCTGACT	179
	R：ATCCGGAGGACTCATTTTCTG
β-actin^b	F：CTGACCCTGAAGTACCCCATT	168
	R：GGTCATCTTCTCACGGTTGG

细粒棘球绦虫钙网蛋白的原核表达及功能初步鉴定

Prokaryotic expression and preliminary functional identification of Echinococcus granulosus calreticulin

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