寄生虫与医学昆虫学报
寄生蟲與醫學昆蟲學報
기생충여의학곤충학보
ACTA PARASITOLOGICA ET MEDICA ENTOMOLOGICA SINICA
2014年
4期
233-239
,共7页
秦梅%汤新明%刘贤勇%陶鸽如%索静霞%田秀玲%索勋
秦梅%湯新明%劉賢勇%陶鴿如%索靜霞%田秀玲%索勛
진매%탕신명%류현용%도합여%색정하%전수령%색훈
和缓艾美耳球虫%转染%黄色荧光蛋白%H9N2禽流感病毒
和緩艾美耳毬蟲%轉染%黃色熒光蛋白%H9N2禽流感病毒
화완애미이구충%전염%황색형광단백%H9N2금류감병독
Eimeria mitis%Stable transfection%Yellow fluorescent protein (YFP)%H9N2 influenza virus
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。
H9N2亞型禽流感病毒已經被證實可以傳播到人類,對它存在的潛在危害已受到越來越多的關註。因此,研究安全而有效的H9N2禽流感病毒疫苗已迫在眉睫。為研究和緩艾美耳毬蟲作為活載體錶達及運輸禽流感病毒抗原的潛能,本研究構建瞭可穩定錶達H9N2禽流感病毒HA1蛋白的轉基因和緩艾美耳毬蟲。利用黃色熒光蛋白(YFP)及融閤的乙胺嘧啶抗性基因(DHFR-TSm2m3)作為報告基因及篩選基因,我們將錶達H9N2禽流感病毒HA1抗原的質粒載體覈轉毬蟲子孢子,接種鷄後在乙胺嘧啶藥物的選擇壓力下進行篩選併連續傳代穫得轉基因和緩艾美耳毬蟲繫。利用染色體步移和免疫印跡證實瞭HA1基因的成功插入和錶達;利用間接免疫熒光證實HA1蛋白定位于毬蟲子孢子細胞膜錶麵和頭部。研究進一步髮現,轉基因毬蟲的繁殖力與野生毬蟲相噹,感染後亦于第6d達到排卵囊高峰。該轉基因和緩艾美耳毬蟲株具有作為疫苗活載體的潛能。
H9N2아형금류감병독이경피증실가이전파도인류,대타존재적잠재위해이수도월래월다적관주。인차,연구안전이유효적H9N2금류감병독역묘이박재미첩。위연구화완애미이구충작위활재체표체급운수금류감병독항원적잠능,본연구구건료가은정표체H9N2금류감병독HA1단백적전기인화완애미이구충。이용황색형광단백(YFP)급융합적을알밀정항성기인(DHFR-TSm2m3)작위보고기인급사선기인,아문장표체H9N2금류감병독HA1항원적질립재체핵전구충자포자,접충계후재을알밀정약물적선택압력하진행사선병련속전대획득전기인화완애미이구충계。이용염색체보이화면역인적증실료HA1기인적성공삽입화표체;이용간접면역형광증실HA1단백정위우구충자포자세포막표면화두부。연구진일보발현,전기인구충적번식력여야생구충상당,감염후역우제6d체도배란낭고봉。해전기인화완애미이구충주구유작위역묘활재체적잠능。
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.