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

H9N2亚型禽流感病毒已经被证实可以传播到人类，对它存在的潜在危害已受到越来越多的关注。因此，研究安全而有效的H9N2禽流感病毒疫苗已迫在眉睫。为研究和缓艾美耳球虫作为活载体表达及运输禽流感病毒抗原的潜能，本研究构建了可稳定表达H9N2禽流感病毒HA1蛋白的转基因和缓艾美耳球虫。利用黄色荧光蛋白（YFP）及融合的乙胺嘧啶抗性基因（DHFR-TSm2m3）作为报告基因及筛选基因，我们将表达H9N2禽流感病毒HA1抗原的质粒载体核转球虫子孢子，接种鸡后在乙胺嘧啶药物的选择压力下进行筛选并连续传代获得转基因和缓艾美耳球虫系。利用染色体步移和免疫印迹证实了HA1基因的成功插入和表达；利用间接免疫荧光证实HA1蛋白定位于球虫子孢子细胞膜表面和头部。研究进一步发现，转基因球虫的繁殖力与野生球虫相当，感染后亦于第6d达到排卵囊高峰。该转基因和缓艾美耳球虫株具有作为疫苗活载体的潜能。

CONSTRUCTION AND IDENTIFICATION OF TRANSGENIC EIMERIA MITIS EXPRESSING HA1 PROTEIN OF H9N2 INFLUENZA VIRUS

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.