成纤维细胞生长因子受体3腺病毒载体的包装与鉴定

Construction and identification of recombinant adenovirus encoding mouse FGFR3 cDNA

目的构建成纤维细胞生长因子受体3(fibroblast growth factor receptors3,FGFR3)腺病毒载体,并进行鉴定、包装及表达检测。方法将小鼠FGFR3cDNA亚克隆至穿梭质粒pAdTrack-CMV上,通过电击转化使其在BJ5183细菌中与AdEasy-1同源重组,通过酶切及测序筛选阳性重组子。以脂质体介导法转染HEK293细胞进行包装、扩增并测定其滴度;并用RT-PCR及Western blot检测FGFR3在HT29细胞中的表达。结果经限制性内切酶检测、测序鉴定和细胞转染后GFP的表达等证实成功地构建了携带FGFR3cDNA的重组腺病毒载体并制备出有感染能力的高滴度重组腺病毒。将构建的腺病毒重组子转染HT29细胞,RT-PCR及Western blot均检测到FGFR3的表达。结论成功地构建了含有FGFR3cDNA的重组腺病毒载体并在HT29中表达了小鼠FGFR3分子,为进一步研究FGFR3的功能及其在骨折愈合中的作用提供有效的基因转移载体。

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 recombinarit adenovirus. The viral titer was 3 ×10^9pfu/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.