Gene therapy DNA vector VTvaf17, production method； E. coli strain SCS110-AF, production method； E. coli strain SCS110-AF/VTvaf17, with gene therapy DNA vector VTvaf17, production method
셀 앤드 진 테라피 엘티디;오브체스트보 에스 오그라니첸노이 오트베트스트벤노스티유 "프로리브니예 이노베이셔니예 테크놀로지"
发明人:
사벨리예바, 나탈리아
申请号：
KR1020207008725
公开号：
KR1020200074944A
申请日:
2018.03.26
申请国别(地区):
KR
年份:
2020
代理人:
摘要：
The present invention relates to genetic engineering and can be used in biotechnology, medicine and agriculture. The present invention provides nucleotide sequence SEQ ID No. There is a need to prepare a 3165-bp gene therapy DNA vector VTvaf17 for genetic modification of animal and human cells containing 1. The method for preparing the 3165-bp gene therapy DNA vector VTvaf17 is, first, a site for the 1188-bp promoter region of human elongation factor EF1A having an endogenous enhancer, restriction endonucleases BamHI, EcoRV, SalI, HindIII, KpnI, EcoRI. Having 35-bp polylinker, 466-bp transcription terminator and polyadenylation sequence of human growth factor, 136-bp regulatory element RNA-OUT of transposon Tn10 to enable abiotic positive selection, cells of most E. coli strains The step of preparing a 4182-bp vector containing a 1299-bp origin of replication, a 1010-bp kanamycin resistance gene for self-replication with single nucleotide substitution to increase vector production in the 4182-bp vector It is cut by the SpeI restriction site, and the remaining pieces are ligated to itself. Certain objectives are achieved by obtaining an E. coli strain SCS110-AF for the production of a gene therapy DNA vector VTvaf17 or gene therapy DNA vector based on the gene therapy DNA vector VTvaf17, which allows for an antibiotic-free positive selection. The method for obtaining the E. coli strain SCS110-AF for the production of a gene therapy DNA vector VTvaf17 or a gene therapy DNA vector based on the gene therapy DNA vector VTvaf17, is a regulatory element RNA of transposon Tn10 that enables a non-antibiotic positive selection. IN, a 1422-bp levansucase gene sacB whose product ensures selection in a sucrose-containing medium, a 763-bp chloramphenicol resistance gene catR required for selection of strain clones with homologous recombination, and gene inactivation A step of preparing a 64-bp linear DNA fragment containing 329-bp and 233-bp, two homologous sequences that guarant
