Construction and identification of recombinant adenovirus encoding mouse FGFR3 cDNA
- VernacularTitle:成纤维细胞生长因子受体3腺病毒载体的包装与鉴定
- Author:
Zhijun LIU
;
Lin CHEN
- Publication Type:Journal Article
- Keywords:
FGFR3;
adenovirus;
transfection
- From:Journal of Third Military Medical University
2003;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant adenovirus encoding mouse wild type FGFR3 cDNA. Methods Mouse FGFR3 cDNA obtained from MoFR3/SV was subcloned into plasmid pBluescript KS and further cloned into plasmid pAdTrack-CMV. The plasmid (pAdTR3) was transferred into BJ5183 cells which contained the adenovirus plasmid (pAdeasy-1) to produce recombinant adenovirus vector encoding FGFR3 cDNA (pAdE-FGFR3). The recombinant adenovirus vector was identified and transfected into the adenoviral packaging cell HEK293 by lipofectamine 2000 to get recombinant adenovirus particles. The adenovirus was confirmed by polymerase chain reactin (PCR) and its titer was determined. Then the recombinant vector was transfected into HT29 cells. The expression of FGFR3 mRNA and protein in HT29 cells was assayed by RT-PCR and Western blotting. Results The recombinant adenovirus vector encoding FGFR3 cDNA was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. The transfected HEK 293 cells were lysed by freeze-thawing to obtain the recombinant adenovirus in the lysate. Further, PCR product of the lysate confirmed the presence of recombinant adenovirus. The viral titer was 3?109pfu/ml. RT-PCR and Western blotting showed that pAdE-FGFR3 transfected HT29 group expressed FGFR3 higher than that of GFP controlled group. Conclusion Infective recombinant adenovirus encoding FGFR3 cDNA was successfully obtained by plasmid homogenous recombination in bacteria, and highly expressed in HT29 cells after transfection, which paves a way for studying the effect of FGFR3 in bone fracture healing.
