果蝇唾液腺特异基因CG8419多克隆抗体的制备及检测

Preparation and Detection of Polyclonal Antibody CG8419 of Specific Gene in Drosophila Salivary Gland

果蝇CG8419基因与脊椎动物中TRIM45基因为同源基因.以果蝇cDNA为模板通过PCR扩增出亲水性和特异性好的果蝇CG8419基因片段,将其克隆入表达载体PET-28a,构建出重组表达质粒PET-28a-CG8419.将重组质粒转化大肠杆菌Rosetta,经IPTG诱导出带His标签的重组融合蛋白.通过尿素洗涤包涵体并切胶回收纯化融合蛋白,然后再免疫新西兰大白兔制备多克隆抗体.Western blot实验分别验证抗体的效价和特异性.果蝇胚胎抗体染色显示该基因在果蝇唾液腺中表达.

CG8419 in Drosophila is the ortholog of TRIM45 in vertebrates.The hydrophilic and well specific fragment of Drosophila CG8419 was obtained with PCR technology.Then it was cloned into the expression vector PET-28a to acquire the recombinant expression plasmid PET-28a-CG8419.It was transformed into Escherichia coli Rosetta,and His-fusion protein was induced with IPTG.The fusion protein was purified by UREA and separated by SDS-PAGE and recovered by gel extraction.White rabbits originate in New Zealand were immunized with the separated protein,the antibody was detected by Western blot.The antibody staining embryos in Drosophila demonstrate the gene expression in the salivary glands.