[Construction and expression of recombinant adenovirus vector Ad5-hTRX-EGFP].
This study was purposed to construct and prepare the recombinant adenovirus vector carrying human thioredoxin (hTRX) and enhanced green fluorescence protein (EGFP), and transfect it into HEK293 cells, so as to lay a foundation for further gene therapy. The PCR-amplified products of hTRX with a pair of primers containing Not I and EcoR V restriction sites were subcloned into shuttle plasmid pDC316-mCMV. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC316-hTRX-EGFP and large adenovirus-helper plasmid pBHGlox (delta) E1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus pAd-hTRX-EGFP was co-transfected in HEK293 cells, purified by CsCl gradient centrifugation, counted for virus particles and determined for titer. The recombinant adenovirus was identified by PCR. The HEK293 cells were then transfected with adenoviruses and assayed by flow cytometry. The expression of hTRX was confirmed by Western blot. The results showed that according to PCR and restriction endonuclease assay, the target gene was inserted into recombinant adenovirus vector successfully. The sequence of fusion gene was the same as that of designed fragments. The titer of the purified recombinant adenovirus pAd-hTRX-EGFP was 5.558×10(10) pfu/ml. A transfection efficiency of 92.25% could be achieved at MOI = 100. Western blot further confirmed that hTRX was efficiently expressed in HEK293 cells. It is concluded that recombinant adenovirus vector containing hTRX has been constructed successfully and obtained highly efficient virus that can express efficiently in HEK293 cells, which laid a foundation for further investigation.
Department of Hematopoietic Stem Cell Transplantation, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, China.
Jiang-Wei
JW
Hu
Jun
J
Wang
Man
M
Xu
Yong-Feng
YF
Shu
Wei-Xia
WX
Kong
Hong-Xia
HX
Sheng
Bin
B
Zhang
Hu
H
Chen
D000256:Adenoviridae / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics*; D014162:Transfection
D011994:Recombinant Proteins; C491203:TXN protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013879:Thioredoxins