摘要
采用显微注射法，将构建的绵羊金属疏因（ｏＭＴ）基因启动子与抗猪瘟病毒核酶（ＨＣＶ－Ｒｉｂｏｚｙｍｅ．ＨＲ）基因融合的质粒ｐＭＨＲ＿（３２）线性ＤＮＡ分子约２００～４００个基因拷贝导入兔原核胚的雄原核中，１５９枚导入基因胚移植给９只受体兔，产仔２２只，移植成活率为１３．８％。２２只仔兔经聚合酶链式反应（ＰＣＲ）和核酸探针杂交检测，６只体内整合有外源目的基因，整合率为２７．３％。再用１００个半数反应量（ＲＩＤ＿（５０））的猪瘟病毒Ｃ系兔化弱毒兔体攻毒，４只外源基因整合的实验兔能完全或部分抵抗ＨＣＶ弱毒攻击，不产生或无明显发热反应；而对照兔和６只ＨＲ基因未整合的实验兔不能抵抗ＨＣＶ弱毒攻击，出现特征性的稽留热型。逆转录聚合酶链式反应（ＲＴ－ＰＣＲ）和ＲＮＡ探针杂交检测４只转基因兔，两只肝中表达出ｏＭＴ－ＨＲｍＲＮＡ。
About 200～400 copies of linear DNA of a recombinant plasmid pMHR32,contai-ning the ovine metallothionein(oMT)gene promoter region joined to a synthetic ribo-zyme gene targeted to the genomic RNA of hog cholera virus(HCV),was introducedinto the pronuclei of 215 feritilized rabbit eggs from 10 donors by microinjection,14%(29/15g)of gene-microinjecting eggs(GMIE)developed to neonates by implatating theminto the reprodl1ctive tracts of 9 pesudopregnant rabbits.27%(6/22)of the neonatesintegrated the foreign oMT-HCV Ribozyme(HR)fusion gene detected by PCR and hyb-ridization with labbeling nucleic acid probes.After infection of attenuated HCV Chinesestrain with 100 RID50,one transgenic rabbit showed normal body temperature,anothershowed temporal increasing body temperature less than 0.5℃ and other two,less than1℃ whereas six experimental rabbits without integration of foreign HR gene and tencontrol rabbits showed characteristic continued fever.50%(2/4)of transgenic rabbitsexpressed oMT-HR gene mRNA in their livers detected by reverse transcription-polyme-rase chain reaction(RTPCR)and hybridization with a RNA probe. These results demon-strated that the foreign oMT-HR gene was expressed in the body of transgenic rabbitsand played a key role in the resistance against infection of attenuated HCV.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
1996年第4期319-325,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家863项目资助课题
关键词
兔
基因转移
猪瘟病毒
核酶
整合与表达
Rabbit,Gene transfer,Hog cholera virus,Ribozyme,Integration andexpression
