Fu Xin, Zou Dong-ting, Zhou Wen-qu, Xing Fu-qi, Li Bing
Experimental Medical Research Center, Guangzhou Medical College, Guangzhou 510182, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Sep;23(9):856-8.
To induce the expression of F10 in E.coli and prepare the rabbit polyclonal antibody against it.
The gene of F10 was amplified by PCR, and cloned into expression vector pET-GST to construct recombinant expression plasmid pET-GST/F10. The recombinant plasmid was transformed into E.coli BL21 and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and analyzed by SDS-PAGE and Western blot. A rabbit was immunized with the purified F10 fusion protein to produce polyclonal antibody, and the production of antibody was confirmed by ELISA.
Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained correct coding region of F10. SDS-PAGE demonstrated that the recombinant protein was expressed with the expected molecular weight at 61 kD. After purified, the purity of the fusion protein was above 90%. Western blot confirmed the recombinant protein was GST/F10 fusion protein. Rabbit polyclonal antibody was obtained, the titer of which was 1:20 000.
F10 recombinant expression vector has been successfully constructed and F10 protein has been expressed. The obtained rabbit anti F10 antibody has a high titer and will facilitate the study of the biological function of F10.
在大肠杆菌中诱导F10表达并制备抗F10兔多克隆抗体。
通过PCR扩增F10基因，克隆至表达载体pET-GST构建重组表达质粒pET-GST/F10。将重组质粒转化至大肠杆菌BL21中，用IPTG诱导表达重组蛋白。融合蛋白经亲和层析进一步纯化，进行SDS-PAGE和Western印迹分析。用纯化的F10融合蛋白免疫家兔制备多克隆抗体，通过ELISA法检测抗体产生情况。
限制性内切酶酶切及DNA测序分析表明重组表达质粒含有正确的F10编码区。SDS-PAGE显示重组蛋白以预期的61 kD分子量表达。纯化后，融合蛋白纯度高于90%。Western印迹证实重组蛋白为GST/F10融合蛋白。获得兔多克隆抗体，其效价为1:20 000。
成功构建F10重组表达载体并表达了F10蛋白。获得的兔抗F10抗体效价高，将有助于F10生物学功能的研究。
