鸡成纤维细胞生长因子Ⅰ型受体胞外段原核表达载体的构建及表达鉴定

Construction and Identification of Recombinant Plasmid Encoding Extracellular Domain of FGFR-1 in Chicken

目的构建重组原核表达载体以获得鸡成纤维细胞生长因子Ⅰ型受体(FGFR-1)胞外段活性蛋白,为进一步研制FGFR-1蛋白质肿瘤疫苗打下基础。方法利用RT-PCR技术,从鸡胚肝中扩增出鸡的FGFR-1胞外段基因的cD-NA,用内切酶消化后插入原核表达质粒pQE30中。经过琼脂糖凝胶电泳和测序鉴定后,体外转化大肠埃希菌,用RT-PCR和免疫印迹方法鉴定是否能够正确表达,同时利用Ni-NTA琼脂柱层析法纯化复性重组蛋白。结果琼脂糖凝胶电泳和测序鉴定证实PCR扩增的cDNA及构建的重组原核表达质粒pQE30-FGFR-1正确,并在大肠埃希菌中正确表达。经纯化后,进行SDS-PAGE电泳显示得到1条分子量约40 ku的蛋白质条带。结论成功构建鸡FGFR-1胞外段原核表达载体并获得重组活性蛋白。

Objective To construct a recombinant prokaryotic expression vector pQE30-FGFR-1 for obtaining the extracellular domain of chicken FGFR-1 protein expressed in E. coli JM109 and developing the genetic engineering tumor vaccine. Methods The cDNAs of extracellular domain of chicken FGFR-1 were amplified by RT-PCR. The cDNAs were digested by restriction endonuclease and inserted into the prokaryotic-expression vector pQE30. The resultant recombinant plasmids were confirmed by agarose gel electrophoresis analysis and sequencing, then transformed into the E. coli JM109. RT-PCR and Western blot were used to certify whether the target proteins were correctly expressed in the E. coli JM109, and Ni-NTA agarose collumn chromatography was used to purify and refold the FGFR-1 protein. Results The cDNAs of the chicken FGFR-1 were correctly amplified. The recombinant prokaryotic plasmids were successfully constructed, and they could be correctly expressed in the E. coli JM109. After purified, the expression product of the plasmids showed an about 40 ku single band on SDS-PAGE. Conclusion The construction of the recombinant prokaryotic plasmids and the preparation of the active protein of chicken FGFR-1 laid a solid foundation for further study on the tumor vaccine.