CONSTRUCTION AND IDENTIFICATION OF TRANSGENIC EIMERIA MITIS EXPRESSING HA1 PROTEIN OF H9N2 INFLUENZA VIRUS
10.3969/j.issn.1005-0507.2014.04.003
- VernacularTitle:表达H9N2禽流感病毒HA1蛋白的转基因和缓艾美耳球虫的构建
- Author:
Mei QIN
1
;
Xin-Ming TANG
;
Xian-Yong LIU
;
Ge-Ru TAO
;
Jing-Xia SUO
;
Xiu-Ling TIAN
;
Xun SUO
Author Information
1. 中国农业大学动物医学院,北京100193
- Keywords:
Eimeria mitis;
Stable transfection;
Yellow fluorescent protein (YFP);
H9N2 influenza virus
- From:
Acta Parasitologica et Medica Entomologica Sinica
2014;(4):233-239
- CountryChina
- Language:Chinese
-
Abstract:
The H9N2 avian influenza virus ( AIV) has become increasingly concerning due to its role in severe economic losses in the poultry industry.Transmission of AIV to mammals, including pigs and humans, has accelerated to devise preventive strategies.To investigate the potential to be used as a vaccine vector for Eimeria mitis expressing antigens from H9N2 AIV, we here successfully developed stable transgenic E.mitis expressing HA1 protein from H9N2 AIV.Using the double-cassette expressing vector strategy with one cassette expressing yellow fluorescent protein ( YFP ) fused to muted dihydrofolate reductasethymidylate synthase derived from Toxoplasma gondii (TgDHFR—TSm2m3), the other expressing HA1 of the H9N2 virus, one transgenic E.mitis population ( Emi. HA1 ) was constructed.Sporozoites of E.mitis transfected with yellow fluorescent protein ( YFP) expression plasmid were inoculated into chickens via the cloacal route. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting ( FACS) and then successively passaged in chickens.The resulting population was analyzed by genome walking, western blot and indirect fluorescent assay ( IFA) . Genome walking confirmed the random integration of plasmid DNA into the genome; while western blot analysis demonstrated the expression of foreign protein-HA1.IFA result indicated the expressed by E.mitis mainly distributed the surface of cell membrane and the head of the sporozoites.We found that the reproduction of Emi.HA1 was similar with that of the parental strain.The expression of foreign antigens in the transgenic parasites will facilitate the development of transgenic E.mitis as a vaccine vector.
