摘要
将人酪氨酸羟化酶cDNA表达盒克隆于质粒型腺病毒载体p△Elsp1A，得到重组质粒pAd－TH。随后用脂质体法将重组质粒pAd－TH和拯救型腺病毒质粒pBHG11一起共转染293细胞，通过体内同源重组生成重组腺病毒AdCMVth，THcDNA重组进入腺病毒E1区并受CMV启动子控制。采用形态学、病毒核酸酶切和PCR／RT－PCR等方法进行鉴定正确。重组腺病毒滴度达到1010pfu／ml。初步结果表明，该重组腺病毒感染MN9D细胞后可使细胞内多巴胺水平增加1倍，显示出明显的TH生物学活性。提示TH重组腺病毒AdCMVth可作为高效的基因转移载体用于帕金森氏病基因治疗。
The human tyrosine hydroxylase(TH) cDNA containing expression cassette was inserted into adenovirus plasmid-type vector of p△Elsp1A, to generate a recombinant plasmid pAd-TH as transfer vector. The plasmid pAd-TH together with adenovirus rescue-type plasmid of pBHG11, were cotransfected into human cell 293 using liposome method, and then recombinant adenovirus AdCMVth was propagated in cell 293 via homologous recombination. The TH cDNA in the adenovirus of AdCMVth was inserted into the early 1 region in the adenovirus genome and driven by the human cytomegalovirus gene promoter (CMV).A series of methods were employed to identify the obtained adenovirus. The titer of virus stocks were generally up to 1010 plaque forming units per milliliter. Our primary results also showed an one-fold increase of dopamine level in MN9D cells infected with AdCMVth, suggesting that AdCMVth provides TH activities in cell. It is concluded that the recombinant adenovirus of AdCMVth with the biological activities might be used as a gene transfer vector in the studies of gene therapy for Parkinson's disease.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
1998年第4期313-317,共5页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金
关键词
酪氨酸羟化酶
重组腺病毒
基因治疗
帕金森氏病
tyrosine hydroxylase, adenovirus, homologous recombination, gene therapy
