Construction and identification of polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS.
OBJECTIVE
To construct the Fas ligand (FasL) and inducible nitric oxide synthase (iNOS) coexpressed PCA13 plasmid used for packing of adenovirus, so as to observe the immunoprotective effect of FasL on the adenovirus vector.


METHODS
By way of the internal ribosome entry site (IRES), gene engineering techniques such as preparation and transformation of competent cells, plasmid extraction, agarose gel electrophoresis and restriction enzymolysis were used for the construction of the polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS, which could coexpress FasL and the iNOS gene after multisubcloning steps.


RESULTS
FasL and iNOS connected with the IRES were successfully cloned to the PCA13 plasmid and verified by enzymolysis (600 bp FasL, 1000 bp IRES and 4000 bp iNOS) and the gene sequence was concordant with the gene bank.


CONCLUSIONS
The polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS was successfully constructed.
Qiu
Jiang Feng
JF
Department of Surgery, Renji Hospital, Shanghai Second Medical University, Shanghai, China. zzq72@sina.com.cn
Wu
Zhi Yong
ZY
Luo
Chun Xia
CX
Yu
Fu Rong
FR
Zhang
Zhi Qi
ZQ
D000256:Adenoviridae / Q000235:genetics* / Q000276:immunology; D053222:Fas Ligand Protein; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006973:Hypertension / Q000628:therapy; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D010957:Plasmids / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics*
D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D015678:Viral Structural Proteins; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II