[Construction of the recombinant adenovirus carrying porcine interferon gamma (poIFNgamma) and identification of its antiviral activity].
The total RNA was extracted from peripheral blood mononuclear cells (PBMC) which was isolated from Meishan porcine and induced with concanavaline A (ConA), then the porcine interferon gamma gene (PoIFNgamma, 501bp) was amplified by RT-PCR. The result of sequencing demonstrated that the amplified PoIFNgamma had 100% nucleotide homology with the other porcine IFNgamma sequence published on GenBank. The objective gene (PoIFNgamma) was inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNgamma. And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37 degrees C for 24h on kanamycin resistance plate and selected for smaller colonies. Then, the extracted recombinant plasmid was named pAd-Sh-PoIFNgamma, which was confirmed by Pac I digestion, and transformed into XL10-Glod(r) for copious preparation. pAd-Sh-PoIFNgamma linearized with Pac I was co-transfected with liposome into 293 package cell-line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome (deleted with E1 and E3 genes) carrying PoIFNgamma was successfully packaged into intact virion. The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR. The antiviral activity of PoIFNgamma was tested by CPE50 method. The results showed that the PoIFNgamma expressed by adenovirus had high antiviral activity, which was 1.3 x 10(6) U/mL against VSV in MDBK cells. The results demonstrated that the recombinant adenovirus carrying PoIFNgamma could be stably passaged.
Yao
Qing-Xia
QX
Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. yaoqingxia@sohu.com
Xu
Zhuo-Fei
ZF
He
Yan-Nan
YN
Si
You-Hui
YH
Qian
Ping
P
Chen
Huan-Chun
HC
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D007371:Interferon-gamma / Q000235:genetics* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D013552:Swine
D000998:Antiviral Agents; D011994:Recombinant Proteins; D007371:Interferon-gamma