学术
猪瘟病毒表面抗原E2基因植物表达载体的构建
祁喜涛[1] 李国强[1] 赵文亮[1] 樊福好[2] 李余良[1] 胡建广[1]
[1]广东省农作物遗传改良重点实验室,广东广州510640 [2]农业部种猪质量监督检验测试中心,广东广州510000
摘 要:
目的：克隆猪瘟病毒表面抗原E2基因,构建种子特异表达的载体,为在玉米种子内专一表达E2蛋白奠定基础。方法：终止序列NOS经中间载体pUC18插入植物表达载体pCAMBIA-1300的XbaⅠ与EcoRⅠ位点间,得重组质粒pCAMBIA-1300-NOS,命名为pCAMBIA-1300-NOS-bar;将1.4kb的种子特异表达启动子Globulin-1插入质粒pCRR○2.1的HindⅢ与BamHⅠ位点间,得重组质粒pCR-G;将1.1kb的E2基因插入质粒pBluescriptSKⅡ的HindⅢ与BamHⅠ位点间,消除HindⅢ位点,得重组质粒pBluescriptSKⅡ-E2;用BamHⅠ和XhoⅠ从pBluescriptSKⅡ-E2中分离E2,插入pCR-G的BamHⅠ和XhoⅠ之间,得重组质粒pCR-G-E2;用HindⅢ和XbaⅠ从pCR-G-E2中分离2.5kb的片断,插入载体pCAMBIA-1300-NOS-bar中,得玉米种子特异表达载体pCAMBIA-1300-GEN。结果：对重组质粒pCAMBIA-1300-GEN经酶切、测序鉴定正确无误。结论：成功构建了种子特异表达载体并将其导入根癌农杆菌LBA4404,为在种子内特异表达猪瘟主要抗原蛋白的转基因植物新品种奠定了基础。[著者文摘]
文章出处:
《生物技术》-2007年17卷5期 -17-20页
分 类 号:
文献标识码:
A
文章编号:
1004-311X（2007）05-0017-04
Construction of A Endosperm - specific Expression Vector Controlling the Gene of Classical Swine Fever Virus Surface Antigen E2
QI xi - tao , LI Guo - qiang , ZHAO Wen - liang, FAN Fu - hao, LI Yu - liang , HU Jian - guang （1. Guangdong Key Laboratory For Crop Genetic Improvement, Guangzhou 510640, China; 2. Center of Quality Test and Smpervision for Breeding Swine Guangzhou,MOA, Guangzhou 510000,China）
Abstract:
To construct a endosperm - specific expression plasmid for expressing the gene of Classical Swine Fever Virus surface antigen E2 in maize endosperm. Methods： The NOS terminator digested by Sac Ⅰ and EcoR I from pGFP- 2 were inserted into the Sac I/EcoR Ⅰsite of pUC18, developed pUC18 - NOS plasmid, the fragments digested by Xba Ⅰ and EcoR Ⅰfrom pUC18 - NOS were then inserted into the Xba Ⅰ / EcoR 1 site of the plasmid pCAMBIA - 1300 through a serial of enzyme digestion and ligation reaction, formed pCAMBIA - 1300 - NOS - bar. Maize endosperm- specific promoter globulin was inserted into the HindUI/BamH Ⅰ site of pCR2.1 to form pCR- G. The E2 gene was inserted into the Hind UI/BamH 1 site of pBluescriptSK Ⅱ resulting in pBluescriptSK Ⅱ - E2, then the Hind UI restriction point was eliminated. E2 gene was separated from the pBluescriptSK Ⅱ - E2 by BamH 1 and Xho I digestion and then inserted into the globulin downstream position of pCR - G resulting in pCR- G- E2. The fragments containing ＂globulin＋ E2＂ digested by HindUI and XhaI from the pCR- G- E2 were inserted into plasmid pCAMBIA - 1300 - NOS - bar, and developed pCAMBIA - 1300 - GEN. Results： Restriction endonuclease digestion and sequence anal- ysis verified successful construction of the recombinant vector pCAMBIA- 1300- GEN. Conclusion： The pCAMBIA- 1300- GEN was directly introduced into Agrobacterium tumefaciens strain LBA4404 and part of transgenic plants have been obtained by Agrobacterium - mediated transformation of maize immature embryos.[著者文摘]
Key words:
vector construction; Classical Swine Fever; seed - specific expression
基金资助:
广东省科技攻关重大专项和广东省国际科技合作项目共同资助（“保护性抗原基因甜玉米种子特异表达研究”,2006A20101006;2005B50201001）
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