Title: Construction and identification of rat mitochondria fusion protein gene recombinant adenovirus vector
Abstract: Objective To construct and identify the recombinant adenovirus carrying rat mitochondria fusion (rMfn2) gene,providing a foundation for further research.Methods With a pair of primers containing Not Ⅰ and Hind Ⅲ restriction sites,the polymerase chain reaction (PCR)-amplified products of rMfn2 were subcloned into shuttle plasmid to obtain pDC316-mCMV-enhanced green fluorescent protein (EGFP)-CMV-rMfn2.The constructed recombinant plasmid was identified by sequencing and used for homologous recombination with adlenovirus backbone plasmid pAd5 in competent cells JM109.HEK293 cells were transfected withthe acquired recombinant adenovirus AD-rMfn2 for packaging,propagation and purification,and the virus titer was determined.Teverse transcriptase (RT)-PCR and Western blotting were used to detect the transcription and expression of rMfn2 respectively.Results PCR,restriction analysis and sequencing validated that adenovirus shuttle plasmid pDC316-mCMV-EGFP-CMV-rMfn2 was constructed correctly.rMfn2 was cloned into adenovirus backbone plasmid pAd5 correctly.After packaging and propagation in HEK293 cells,recombinant adenovirus with a titer of 4 × 1013 TU/L was obtained.The transcription and expression of rMfn2 gene were proved in HEK293 cells after infection with the recombinant adenovirus in 48 h.Conclusion Recombinant adenovirus vector containing rMfn2 has been constructed successfully,which established a basis for further research.
Key words:
Mitochondria fusion protein gene; Recombinant adenovirus
Publication Year: 2014
Publication Date: 2014-02-08
Language: en
Type: article
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