中国兽医杂志2025，Vol.61Issue(10)：9-17,9.DOI:10.20157/j.cnki.zgsyzz.2025.10.002
融合表达RHDV1 P2亚域和兔源C5a的重组早熟大型艾美耳球虫的构建与鉴定
Construction and Identification of a Recombinant Precocious Line of Eimeria magna Expressing Fusion Protein of RHDV1 P2 Subdomain and Rabbit-derived C5a
摘要
Abstract
Complement component C5a,known for its role as an immunomodulatory molecule,has recently been recognized as a promising molecular adjuvant for the development of novel vaccines.To explore the feasibility of using C5a as a molecular adjuvant in Eimeria live vector vaccines,this study amplified the rabbit C5a gene via homologous sequence alignment and reverse transcription polymerase chain reaction(RT-PCR),and performed bioinformatic predictions of its sequence,and secondary and tertiary protein structures.A plasmid containing both the P2 subdomain gene of the VP60 protein from rabbit hemorrhagic disease virus type 1(RHDV1)and the C5a gene was constructed,and transfected into sporozoite nuclei followed by serial in vivo passages in rabbits yielded transgenic precocious line of Eimeria magna lines.The transgenic E.magna were identified by polymerase chain reaction(PCR),Western blot,and indirect immunofluorescence assay(IFA).The rabbit C5a gene was determined to be 222 bp in length,with nucleotide sequence homology of 71.2%-77.0%to human,pig,and bovine C5a,and 68.5%to mouse and rat C5a.Structural predictions indicated that C5a is a monomeric protein primarily composed of four α-helices and contains an anaphylatoxin-like domain.Fluorescent oocysts were obtained in the first generation and passaged to the 15th generation,with the 9th generation showing the highest fluorescence rate(39.5%,meeting the identification threshold).Molecular analyses confirmed successful insertion and expression of the P2-C5a gene,and the fusion protein was mainly localized in the cytoplasm of sporozoites.These results demonstrate the preliminary successful construction of a transgenic precocious line of E.magna expressing the P2-C5a fusion protein,providing a foundation for further studies on the adjuvant effect of C5a in transgenic precocious line of Eimeria vaccine systems.关键词
早熟大型艾美耳球虫/兔出血症病毒1型(RHDV1)/P2亚域/补体C5aKey words
precocious line of Eimeria magna/rabbit hemorrhagic disease virus type 1(RHDV1)/P2 subdomain/comple-ment C5a分类
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陈文轩,索静霞,孔嘉华,葛晓敏,裘鑫珠,汤新明,余芳,索勋,刘贤勇..融合表达RHDV1 P2亚域和兔源C5a的重组早熟大型艾美耳球虫的构建与鉴定[J].中国兽医杂志,2025,61(10):9-17,9.基金项目
国家兔产业体系-兔寄生虫病防控岗位科学家(CARS-43-3) （CARS-43-3）
