Preparation and activity detection in vitro of the recombinant adeno-associated vi-rus of Eimeria stiedai ASP
10.16303/j.cnki.1005-4545.2025.11.12
- VernacularTitle:斯氏艾美耳球虫ASP重组腺相关病毒的制备及体外活性检测
- Author:
Yahuan LI
1
;
Chaofan LI
1
;
Mengge CHEN
1
;
Xin LI
1
;
Xiaocen WANG
1
;
Xu ZHANG
1
;
Pengtao GONG
1
;
Nan ZHANG
1
;
Jianhua LI
1
Author Information
1. 吉林大学动物医学学院人兽共患病研究教育部重点实验室/人畜共患传染病重症诊治全国重点实验室,吉林长春 130062
- Publication Type:Journal Article
- Keywords:
Eimeria stiedai;
ASP;
recombinant adeno-associated virus
- From:
Chinese Journal of Veterinary Science
2025;45(11):2387-2393
- CountryChina
- Language:Chinese
-
Abstract:
To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.
