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

Abstract

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

CLC Number:

S852.734

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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Prokaryotic Expression and Secretion Characterization of Annexin B5, B15, and B25 from Echinococcus granulosus

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