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| Construction and indentification of recombinant adenovirus vector containing mouse IFN-λ2 gene |
| （1.Department of Respiratory Medicine，the Affiliated People′s Hospital of Jiangsu University，Zhenjiang Jiangsu 212002;2.Department of Critical Care Medicine，Huangshi Central Hospital，Huangshi Hubei 435000，China） |
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Abstract Objective: To construct recombinant adenovirus vector containing mouse IFN-λ2 gene. Methods: Full-length of mIFN-λ2 cDNA was obtained by using RT-PCR from cells of mouse spleen stimulated by vesicular stomatovirus and then subcloned to the vector of pShuttle- CMV. Generation of recombinants by cotransforming the PmeⅠ-cut shuttle plasmids with pAdEasy-1 backbone vectors in BJ5183，virus was amplified in 293 cells. RT-PCR was used to identify mIFN-λ2 gene had transformed into recombinant adenovirus，and the method of TCID50 was used to measure the virus titer. The mouse lung adenocarcinoma cells were infected by the recombinant adenovirus，the expression of mIFN-λ2 was detected by Western bolt，and the inhibitory effect of mIFN-λ2 to mouse lung adenocarcinoma cells was observed by inverted microscope. Results: The sequence of mIFN-λ2 gene was identical with that reported in GenBank.The recombinant adenovirus vector was successfully constructed and verified by restriction endonucleases digestion. Recombinant adenovirus was successfully produced and amplified in 293A cell line. The constructed recombinant adenovirus was verified containing mouse IFN-λ2 gene，and its virus titer was 2×1010 pfu/ml. Western bolt indicated mIFN-λ2 protein was expressed in the supernatant of mouse lung adenocarcinoma cells infected by the recombinant adenovirus.The inhibitory effect of mIFN-λ2 to mouse lung adenocarcinoma cells was identified by inverted microscope. Conclusion: The adenovirus vector of mIFN-λ2 was successfully constructed.The mIFN-λ2 protein was expressed on mouse lung adenocarcinoma cells infected by the recombinant adenovirus and the growth of mouse lung adenocarcinoma cells was inhibited. This result lays not only a solid foundation for a further study of the impact of the IFN-λ on tumor but also for gene therapy.
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2012, Vol. 22 
