癌钙调蛋白／鱼精蛋白截短体融合基因在原核表达载体中的构建及鉴定

Construction and identification of OM/tp fusion gene by prokaryotic expression vector

目的在原核表达载体中构建癌钙调蛋白(OM)/鱼精蛋白截短体(tp)融合基因,进行基因测序鉴定。方法提取SD大鼠腹腔巨噬细胞,并进行细胞计数,经巨噬细胞特异性抗体CD68鉴定。设计引物扩增OM基因,并在其3’端引入tp的编码基因,经PCR扩增获得OM/tp融合基因,通过中间载体PUC57进行目的基因克隆,将阳性克隆质粒酶切,与原核表达载体PET32a连接,转化大肠杆菌感受态细胞,筛选阳性克隆,小量提取质粒,送样经公司测序鉴定。结果琼脂糖凝胶电泳获得OM/tp目的基因,与预期的目的基因大小一致,回收目的基因与中间载体PUC57连接转化大肠杆菌感受态细胞DH5α后,菌检PCR获得约500bp的阳性克隆,与预期大小一致。抽提质粒PUC57-OM/tp,经测序鉴定后与预期的序列一致。质粒PUC57-OM/tp酶切后与载体PET32a连接转化大肠杆菌感受态细胞DH5α,菌检PCR获得约500bp的阳性克隆,与预期大小一致。抽提质粒PET32a-OM/tp经测序后与预期的序列一致。结论 OM/tp融合基因的成功构建,为该融合蛋白促进视神经损伤后再生的研究奠定了基础。

Background Oncomodulin（OM） is a kind of micromolecule protein. It was found by Macmanus in the end of 1970s, and was extract from rat＇ s hepatoma tissue. It is certified that OM could promote the generation of nerve, especially to optic nerve. Objective The aim of this study was to construct a fusion gene of OM/tp （ oneomodulin/truncated protamine） with prokaryotic expression vector and validate the gene expression by DNA sequencing. Methods Macrophage was isolated from rats＇ abdominal cavity and its purity was confirmed by maerophages specific antibody CD68. Three oligonueleotide primers were designed and used to amplify the OM gene, and coding sequence of tp was added in the 3 ＇ primer termination. The OM/tp gene was amplified by PCR and cloned into middle expression vector PUC57. The positive ctoned plasmid was digested with restriction endonuclease and connected with prokaryotic expression vector PET32a, and then transformed into E coli DH5α. The positive clone was selected and the plasmid was extracted for sequencing. Results The expected PCR product was revealed by agarose gel electrophoresis. After the cloned gene was connected with PUC57 and transferred to E coli DH5α ,the amplified target band at 500 bp was exhibited with the consistent size with expected one. The sequence was completely matched to the designed sequence. The plasmid PUC57-OM/tp was digested by restriction endonuelease and connected with PET32a. After transformed into E eoli DH5α,the amplified target bands at 500 bp were exhibited with the consistent size with expected one. The gene structure was confirmed by sequence analysis. Conclusion The fusion gene of OM/tp is successfully constructed and identified in this study.