遗传
遺傳
유전
HEREDITAS(BEIJING)
2004年
4期
425-431
,共7页
胡蓉%魏泓%愠律拼%何永睿
鬍蓉%魏泓%慍律拼%何永睿
호용%위홍%온률병%하영예
甲型肝炎病毒衣壳蛋白融合基因%柑橘%植物表达载体%遗传转化%食用疫苗
甲型肝炎病毒衣殼蛋白融閤基因%柑橘%植物錶達載體%遺傳轉化%食用疫苗
갑형간염병독의각단백융합기인%감귤%식물표체재체%유전전화%식용역묘
hepatitis A capsid protein fusion gene%Citrus%plant expression vector%genetic transformation%edible vaccine
基因工程领域的研究进展使得植物体成为具有重要经济价值的药用蛋白的生产体系.以含甲型肝炎病毒结构基因cDNA的克隆载体pCDNAⅡA16为模板,用甲型肝炎病毒衣壳蛋白融合基因特异引物进行PCR扩增,得到全长2.2kb衣壳蛋白融合基因序列.经测序鉴定后正向克隆于植物表达载体pBI121中,衣壳蛋白融合基因位于pBI121质粒T-DNA左右边界区间内,处于CaMV35S启动子控制之下.经限制性内切酶分析和PCR鉴定后利用冻融法将重组质粒pBI121-A导入根癌农杆菌LBA4404.以锦橙 (Citrus. Sinensis Osbeck) 上胚轴为转化材料,通过根癌农杆菌介导法将衣壳蛋白融合基因转化到植物基因组中.120株转化外植体经卡那霉素50 mg/L筛选,其中13株生长状况良好未出现白化现象的拟转化芽微嫁接到实生砧木继续培养.PCR分析证明,13株拟转化植株中有5株植物基因组中已导入甲型肝炎病毒衣壳蛋白融合基因,转化率为4.1%.此研究是对遗传转化柑桔表达外源蛋白的初步探讨,为进一步研究食用疫苗开辟了新途径.
基因工程領域的研究進展使得植物體成為具有重要經濟價值的藥用蛋白的生產體繫.以含甲型肝炎病毒結構基因cDNA的剋隆載體pCDNAⅡA16為模闆,用甲型肝炎病毒衣殼蛋白融閤基因特異引物進行PCR擴增,得到全長2.2kb衣殼蛋白融閤基因序列.經測序鑒定後正嚮剋隆于植物錶達載體pBI121中,衣殼蛋白融閤基因位于pBI121質粒T-DNA左右邊界區間內,處于CaMV35S啟動子控製之下.經限製性內切酶分析和PCR鑒定後利用凍融法將重組質粒pBI121-A導入根癌農桿菌LBA4404.以錦橙 (Citrus. Sinensis Osbeck) 上胚軸為轉化材料,通過根癌農桿菌介導法將衣殼蛋白融閤基因轉化到植物基因組中.120株轉化外植體經卡那黴素50 mg/L篩選,其中13株生長狀況良好未齣現白化現象的擬轉化芽微嫁接到實生砧木繼續培養.PCR分析證明,13株擬轉化植株中有5株植物基因組中已導入甲型肝炎病毒衣殼蛋白融閤基因,轉化率為4.1%.此研究是對遺傳轉化柑桔錶達外源蛋白的初步探討,為進一步研究食用疫苗開闢瞭新途徑.
기인공정영역적연구진전사득식물체성위구유중요경제개치적약용단백적생산체계.이함갑형간염병독결구기인cDNA적극륭재체pCDNAⅡA16위모판,용갑형간염병독의각단백융합기인특이인물진행PCR확증,득도전장2.2kb의각단백융합기인서렬.경측서감정후정향극륭우식물표체재체pBI121중,의각단백융합기인위우pBI121질립T-DNA좌우변계구간내,처우CaMV35S계동자공제지하.경한제성내절매분석화PCR감정후이용동융법장중조질립pBI121-A도입근암농간균LBA4404.이금등 (Citrus. Sinensis Osbeck) 상배축위전화재료,통과근암농간균개도법장의각단백융합기인전화도식물기인조중.120주전화외식체경잡나매소50 mg/L사선,기중13주생장상황량호미출현백화현상적의전화아미가접도실생침목계속배양.PCR분석증명,13주의전화식주중유5주식물기인조중이도입갑형간염병독의각단백융합기인,전화솔위4.1%.차연구시대유전전화감길표체외원단백적초보탐토,위진일보연구식용역묘개벽료신도경.
The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments. 13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.
