细粒棘球绦虫膜联蛋白B5、B15和B25的原核表达和分泌特性分析

doi:10.11843/j.issn.0366-6964.2024.06.032

摘要/Abstract

摘要：

旨在探讨细粒棘球绦虫膜联蛋白B5、B15和B25的反应原性及其在中间宿主组织中的分泌特性，为进一步研究细粒棘球绦虫膜联蛋白与中间宿主的相互作用奠定基础。作者提取细粒棘球蚴原头蚴的总RNA并反转录为cDNA，以cDNA为模板PCR扩增获得EgANXB5、EgANXB15和EgANXB25基因全长编码序列，使用同源重组法构建pET32a-EgANXB5、pET32a-EgANXB15和pET32a-EgANXB25质粒并进行重组蛋白的原核表达，应用蛋白免疫印迹对上述重组蛋白进行鉴定，并采用免疫荧光染色试验分析EgANXB5、EgANXB15和EgANXB25在中间宿主组织中的分泌特性。结果显示：本研究成功克隆并表达出重组EgANXB5、EgANXB15和EgANXB25，并更正了EgANXB25的CDS序列(GenBank：OR245515.1)。蛋白免疫印迹结果显示，这3种蛋白均能够被感染细粒棘球绦虫的犬阳性血清和感染细粒棘球蚴的小鼠阳性血清所识别。同时，免疫荧光染色结果显示，在细粒棘球蚴包囊附近的肝实质组织中可以检测到EgANXB5、EgANXB15和EgANXB25的阳性信号。EgANXB5、EgANXB15和EgANXB25具有良好的反应原性，同时它们具有分泌进入包囊周围肝实质组织中的潜能，可能进一步参与细粒棘球蚴与宿主的相互作用。

关键词: 细粒棘球绦虫, 膜联蛋白, 原核表达, 反应原性, 分泌特性

Abstract:

This study aims to investigate the immunoreactivity and secretion characteristics of the Echinococcus granulosus (E. granulosus) annexin B5, B15, and B25 in the intermediate host tissues, laying the foundation for further research on the interaction between annexin and hosts. In this study, total RNA was extracted from the protoscolex of E. granulosus, and then reverse transcribed into cDNA. The full-length coding sequences of EgANXB5, EgANXB15, and EgANXB25 genes were amplified by PCR using the cDNA as a template. The recombinant plasmids pET32a-EgANXB5, pET32a-EgANXB15, and pET32a-EgANXB25 were constructed using homologous recombination method, and the recombinant proteins were expressed in Escherichia coli. The immunoreactivity of these recombinant proteins was analyzed using Western blotting, and their secretion in the intermediate host tissues was examined by immunofluorescence staining. The recombinant EgANXB5, EgANXB15, and EgANXB25 were successfully cloned and expressed. The coding sequence (CDS) of EgANXB25 was corrected (GenBank: OR245515.1). These three proteins could be specifically recognized by positive canine sera for E. granulosus and positive mouse sera for E. granulosus, indicating that they had strong immunoreactivity. Moreover, immunofluorescence staining revealed that EgANXB5, EgANXB15, and EgANXB25 were primarily distributed in the liver parenchyma near the E. granulosus cyst. Recombinant EgANXB5, EgANXB15, and EgANXB25 exhibite strong immunoreactivity and these proteins have the potential to secrete into the hepatic parenchymal tissue surrounding the cyst, possibly further participating in the interaction between E. granulosus and their host.

Key words: Echinococcus granulosus, annexin, prokaryotic expression, immunoreactivity, secretion characterization

中图分类号:

S852.734

陈延鑫, 华瑞其, 邵国庆, 朱小伟, 侯巍, 李盛琼, 阳爱国, 杨光友. 细粒棘球绦虫膜联蛋白B5、B15和B25的原核表达和分泌特性分析[J]. 畜牧兽医学报, 2024, 55(6): 2607-2618.

Yanxin CHEN, Ruiqi HUA, Guoqing SHAO, Xiaowei ZHU, Wei HOU, Shengqiong LI, Aiguo YANG, Guangyou YANG. Prokaryotic Expression and Secretion Characterization of Annexin B5, B15, and B25 from Echinococcus granulosus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2607-2618.

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基因名称 Gene name	引物序列(5′→3′) Primer sequence
EgANXB5	F: $\underline{{\rm{GCCATGGCTGATATCGGATCC}}}$ATGGTACGAGGGGGGACTGTC
	R: $\underline{{\rm{GCAAGCTTGTCGACGGAGCTCT}}}$CAGCTGTCCGATTCTCGAAGG
EgANXB15	F: $\underline{{\rm{GCCATGGCTGATATCGGATCC}}}$ATGGCGTGCGTTAAACCAGT
	R: $\underline{{\rm{GCAAGCTTGTCGACGGAGCTCT}}}$CAATCCTCATCTTGTTCCAG
EgANXB25	F: $\underline{{\rm{GCCATGGCTGATATCGGATCC}}}$ATGCAGACGGGAGCCACGGTTTTAG
	R: $\underline{{\rm{GCAAGCTTGTCGACGGAGCTCT}}}$CATTTTTCACCAAGTATGGCGAG

免疫接种 Immunization	抗原形式 Antigen form	注射剂量/(mg·只^-1) Injection dose	注射部位 Injection site	注射方式 Injection method
首免 Prime	弗氏完全佐剂+抗原 Freund′s complere adjuvant+Antigen	0.35	背部Back	皮下 Subcutaneous
二免(首免后7 d) The 1st boost (At 7 days post Prime)	弗氏不完全佐剂+抗原 Freund′s incomplete adjuvant+Antigen	0.35	背部Back	皮下 Subcutaneous
三免(首免后14 d) The 2nd boost (At 14 days post Prime)	弗氏不完全佐剂+抗原 Freund′s incomplete adjuvant+Antigen	0.35	背部Back	皮下 Subcutaneous
四免(首免后21 d) The 3rd boost (At 21 days post Prime)	弗氏不完全佐剂+抗原 Freund′s incomplete adjuvant+Antigen	0.35	背部Back	皮下 Subcutaneous

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