Adenovirus-mediated co-expression of the TRAIL and HN genes inhibits growth and induces apoptosis in Marek's disease tumor cell line MSB-1.
BACKGROUND
The objective of this study was to determine the in vitro tumor-inhibitory effect of a recombinant adenovirus expressing a fusion protein of tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) and hemagglutinin-neuraminidase (HN) genes on the MSB-1 Marek's disease tumor cell line.


METHODS
TRAIL and HN genes were amplified from lymphocytes in the peripheral blood of chickens and the LaSota strain of Newcastle disease virus (NDV), respectively, using RT-PCR. The two genes were connected with a 2A connecting peptide by site-directed mutagenesis and gene splicing by overlap extension (SOE). The target gene TRAIL-2A-HN was cloned into the shuttle vector pShuttle-CMV. Homologous recombination was carried out with the vector pAdeasy-1 in the bacterium BJ5183 to construct the recombinant adenovirus plasmid pAd-TRAIL-2A-HN. After linearization, the plasmid was transfected into AD293 cells and packaged. Real-time quantitative PCR (RT-PCR) and fluorescence microscopy confirmed the introduction of the recombinant adenovirus into AD293 cells. The TCID50 method (50% tissue culture infectious dose) was employed to determine viral titers for the exprimental and control viruses, which met criteria for use. The Marek's disease tumor cell line MSB-1 was transfected with the constructed recombinant adenovirus. The infectivity of the recombinant adenovirus and the expression levels of exogenous genes were detected with RT-PCR and western blotting. The effects of the recombinant adenovirus on the growth of MSB-1 cells and cellular apoptosis were determined using flow cytometry.


RESULTS
The recombinant adenovirus infected the cultured cells in vitro, and replicated and expressed exogenous genes in the cells. The recombinant adenovirus Ad-TRAIL-2A-HN inhibited the growth of MSB-1 cells and induced apoptosis by expressing exogenous genes. The rate of induced MSB-1 cell apoptosis reached 11.61%, which indicated that TRAIL and HN produced synergistic tumor-inhibiting effects.


CONCLUSIONS
The constructed TRAIL-2A-HN fusion gene combined the apoptosis-inducing function of TRAIL and the adsorptive capacity of HN from NDV for tumor cells, and the capacity of the recombinant adenovirus expressing this fusion gene to induce tumor cell apoptosis was reported. These results provide a basis for future in vivo tumor suppression studies using recombinant adenoviruses.
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Dongxiao
D
Dong
Tianjing Shenji Group Co., Ltd, Tianjing, 300380 China.
Jing
J
Gao
Tianjing Ruipu Group Co., Ltd, Tianjing, 300380 China.
Ying
Y
Sun
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Yuqing
Y
Long
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Meng
M
Li
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Dongchao
D
Zhang
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Jianfang
J
Gong
Tianjing Ruipu Group Co., Ltd, Tianjing, 300380 China.
Liang
L
Xu
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Liuan
L
Li
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Shunyi
S
Qin
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Jifei
J
Ma
College of Animal Sciences and Veterinary Medicine, Tianjing Agricultural University, Tianjing, 300384 China.
Tianming
T
Jin
Hemagglutinin-neuraminidase; In vitro tumor-inhibiting effect; MSB-1 cell line; Recombinant adenovirus; Tumor necrosis factor (TNF) related apoptosis inducing ligand