CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >

2023 , Vol. 41 >Issue 4: 497 - 501

DOI: https://doi.org/10.12140/j.issn.1000-7423.2023.04.017

SHORT COMMUNICATIONS

Cloning and prokaryotic expression of Theileria equi rhoptry neck protein 5 gene

  • LU Xing ,
  • WANG Shuiyi ,
  • CHEN Linjun ,
  • LIU Mingming ,
  • LIU Yutong ,
  • ZHU Huiru ,
  • JIANG Bingbing ,
  • DU Shaolei ,
  • BAYIN Chahan ,
  • LIU Dandan ,
  • ZHANG Wei
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  • 1 College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
    2 Technology Center, Hohhot Customs District, Hohhot 010020, Inner Mongolia, China

Received date: 2022-11-22

  Revised date: 2023-03-28

  Online published: 2023-09-06

Supported by

Tianshan Youth Program of Xinjiang Uygur Autonomous Region-Outstanding Young Scientific and Technological Talents Project(2020Q014);Xinjiang Agricultural University Post Doctoral Research Mobile Station Funding Project

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Abstract

The rhoptry-neck protein 5 (ron5) gene was PCR amplified from the Xinjiang isolates of Theileria equi. The prokaryotic expression vector of pGEX-4T-ron5 was constructed and transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The expression products was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting analysis was performed using glutathione S-transferase labeled rabbit monoclonal antibody (1:2 000) and horse serum infected with T. equi (1:100) as primary antibodies, and horseradish peroxidase labeled sheep anti-rabbit IgG and rabbit anti-horse IgG (1:5 000) as secondary antibodies. The ron5 of the Xinjiang isolate of T. equi was 1 434 bp, and the entry number obtained by GenBank was OP777492. Sequence comparison results showed that the nucleotide and amino acid sequences of ron5 of the Xinjiang isolate were the most consistent with those of T. equi WA strain (GenBank accession number XM 004833657), which were 99.6% and 99.2%, respectively. Phylogenetic tree analysis showed that the Xinjiang isolate and the T. equi WA strain were clustered on the same branch, with the most closely relation. SDS-PAGE analysis showed that the protein expression was the highest at 3 h induced by isopropyl thiogalactoside. The relative molecular mass (Mr) of RON5 is about 79 000, which is mainly expressed in the form of inclusion bodies. Western blotting results showed that RON5 could be recognized by glutathione S-transferase labeled antibody and horse serum infected with T. equi, with an obvious band at Mr 79 000. In this study, ron5 was successfully cloned, and the RON5 expressed had good antigenicity.

Key words： Theileria equi; Rhoptry-neck protein 5; Prokaryotic expression; Antigenicity

Cite this article

LU Xing , WANG Shuiyi , CHEN Linjun , LIU Mingming , LIU Yutong , ZHU Huiru , JIANG Bingbing , DU Shaolei , BAYIN Chahan , LIU Dandan , ZHANG Wei . Cloning and prokaryotic expression of Theileria equi rhoptry neck protein 5 gene[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023 , 41(4) : 497 -501 . DOI: 10.12140/j.issn.1000-7423.2023.04.017

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