Construction and identification of an adeno-associated viral vector serotype2/9 containing human thyrotropin receptor A subunit

Objective: To construct an adeno-associated viral vector serotype2/9 containing human thyrotropin receptor (rAAV2-9-hTSHR289-IRES-ZsGreen) so as to provide a better means for establishing an ideal animal model and the gene prevention against Graves’ disease. Methods: AAV skeleton plasmid pAAV-IRES-Z...

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Published inXi'an jiao tong da xue xue bao. Journal of Xi'an Jiaotong University (medical sciences). Yi xue ban no. 6; p. 997
Main Authors Feng-yi, ZHAO, Ming-qian, HE, WANG, Yue, ZHANG, Meng, Shi-qian, HU, Bing-yin, SHI, Li-ping, WU
Format Journal Article
LanguageChinese
Published Xi'an Xi'an Jiaotong University 01.01.2019
Subjects
Endocrinology
Gene expression
Gene therapy
Plasmids
Vectors (Biology)
Viruses
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Summary:Objective: To construct an adeno-associated viral vector serotype2/9 containing human thyrotropin receptor (rAAV2-9-hTSHR289-IRES-ZsGreen) so as to provide a better means for establishing an ideal animal model and the gene prevention against Graves’ disease. Methods: AAV skeleton plasmid pAAV-IRES-ZsGreen and PDC315 plasmid containing hTSHR289 were digested by EcoRⅠ+BamHⅠ. The digestion products were connected. And pAAV-hTSHR289-IRES-ZsGreen vector was generated after transformation of JM109 by ligation products. The recombinant plasmid was identified by gel electrophoresis and sequencing. Three plasmids (pAAV-hTSHR289-IRES-ZsGreen, pHelper, and pAAV-2/9) were transfected into 293AAV cell line by calcium phosphate method and a large amount of rAAV2/9-hTSHR289-IRES-ZsGreen was obtained. After purification, the packaging efficiency of recombinant adeno-associated virus was observed by using fluorescence microscope, and its titer was determined by rQ-PCR. HEK293 cells were transfected with rAAV2/9-hTSHR289-IRES-
ISSN:1671-8259
DOI:10.7652/jdyxb201906030