摘要
应用LA-PCR的方法,从荷斯坦牛基因组中成功地扩增获得6.0 kb和1.78 kb两个基因片段,其中6.0 kb包括αs1-酪蛋白基因5′调控序列到第三内含子部分,1.78 kb包括αs1-酪蛋白基因第十二内含子到第十四内含子之间序列,作为打靶载体的同源臂;从新生儿血液基因组中扩增获得5.5 kb的血小板生成素(thrombopoietin,TPO)基因组序列,包括第一内含子部分序列到3′终止子后的序列。以水牛β-酪蛋白基因的5′调控区4.4 kb片段为启动子,以pLoxp质粒为打靶载体骨架,加上同源臂片段和目的基因TPO的片段,经多步酶切-连接-转化-筛选-鉴定基因重组过程,最终建成长达25 kb的人TPO基因定点整合荷斯坦牛αs1-酪蛋白基因打靶载体,PCR和酶切分析表明载体构建正确,为研制定点整合荷斯坦牛乳腺生物反应器奠定了基础。
Using LA-PCR method, two fragments 6.0 kb and 1.78 kb of αs1-casein gene were amplified and cloned from the Holstein cow genome. The 6.0 kb fragment lies between the 5′ flanking region and the third intron, and the 1.78 kb between the twelfth and the fourteenth intron of αs1- cas, ein gene locus. A 5. 5 fragment of thrombopoietin gene （TPO） was cloned from the genome of a new born baby＇s blood, which included the first intron and stop codon of the TPO gene. With the 5′ flanking region of buffalo beta-casein as promoter, the pLoxp plasmid as skeleton, adding the above gene fragment, with multiple steps of restriction enzyme cutting--ligasing--screening--identifing, a gene targeting vector was constructed . The vector was proved to be correct by PCR and restriction enzyme analysis, which was a good start to get the high-level expressing bioreactor.
出处
《广西农业生物科学》
CAS
CSCD
2008年第2期93-98,114,共7页
Journal of Guangxi Agricultural and Biological Science
基金
国家863计划项目(2007AA100505)
广西大学科研基金项目(X071101)
