[Construction and expression of recombinant adenovirus carrying human SHP-1 gene]

Xiao-ling Wang, Sha Zhao, Jian-bo Yu, Rui Huang, Ying-chun Zhang, Wei-ping Liu
Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition 2007, 38 (4): 561-4

OBJECTIVE: To construct and identify recombinant adenovirus Ad-SHP1 carrying human SH2 domain-containing protein tyrosine phosphatase (SHP-1).

METHODS: Human SHP-1 cDNA from healthy volunteers' peripheral blood lymphocyte was cloned into the shuttle plasmid pAdTrack-CMV by standard procedure. The plasmid pAdTrackCMV-SHP1 was selected and then linearized by Pme I, followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183. Recombinant adenovirus Ad-SHP1 were obtained after packaged in HEK293 cells and the green fluorescence in HEK293 cells was observed. Recombinant adenovirus was confirmed with PCR and the expressed SHP-1 protein was verified with Western Blotting. Ad-SHP1 was multiplied and purified. The titer of virus was measured with fluorescent counter. Results The results of sequencing, restriction endounclease digestion and Western Blotting indicated that Ad-SHP1 was successfully constructed. The titer of Ad-SHP1 reached 1.58 x 10(10) pfu/mL.

CONCLUSION: Recombinant adenovirus Ad-SHP1 had been successfully constructed. It could express SHP-1 protein stably and effectively and this will be very helpful for the further study of the generation of extranodal NK/T cell lymphoma and the corresponding therapy.

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