[Construction and identification of recombinant adenovirus expressing rat proteoglycan II].
To construct recombinant adenovirus expressing rat proteoglycan II (Ad-DCN) and to study its biological features.



Rat DCN gene was inserted into an E1 and E3-substituted adenovirus shuttle plasmid. And this shuttle plasmid including DCN was recombined with AdEasy-1 in BJ5183-AD-1 electroporation competent cells to form recombinant adenovirus-DCN plasmid, which was further transfected into Ad293 cells to passage adenovirus. The control recombinant adenovirus, Ad-LacZ, was also constructed in the same way. After plaque forming trail and adjusted with PBS, Ad-DCN and Ad-LacZ were obtained with titer 1x10(9) pfu x ml(-1). PCR, Western blot and MTT analysis were used to detect the expression of DCN or the bioactivity of expressed DCN.



DCN was detected in Ad-DCN infected CHO cells by PCR and Western blot, but not in Ad-LacZ infected CHO cells. MTT analysis results showed that the supernatant from the culture of Ad-DCN infected CHO cells could abrogate the inhibitive effect of TGFbeta1 on proliferation of CCL-64 cells. The proliferation rate of TGFbeta1 + Ad-DCN treated cells was significantly higher than that of TGFbeta1 + Ad-lacz or TGFbeta1 treated cells [(0.5252 +/-0.04 compared with 0.2826 +/-0.02 or 0.2918 +/-0.02) OD, P <0.05] and lower than that of control cells [(0.9332 +/-0.08) OD, P <0.05].



The constructed recombinant adenovirus can express biologically active decorin.
Wu
Fang
F
Department of Endocrinology and Metabolism, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Yao
Hang-ping
HP
Dong
Feng-qin
FQ
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D005822:Genetic Vectors; D008297:Male; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic
D011509:Proteoglycans; D011994:Recombinant Proteins