摘要
目的:克隆人乳珠蛋白(hMAM)编码全长cDNA,原核表达并纯化蛋白产物,为后期研制乳腺癌早期诊断试剂盒奠定基础。方法:自乳腺癌组织及乳腺癌细胞株MD-MB453提取总RNA,通过RT-PCR克隆hMAM cD-NA,构建pQE40-hMAM表达质粒,在大肠杆菌M15中表达,利用镍-亚硝胺乙酸组氨酸(N i-NTA-H is)亲和层析法对重组蛋白进行纯化。结果:乳腺癌组织和乳腺癌细胞株中发现两种hMAM cDNA亚型,即hMAM和hMAM(Isoform),长度分别为279和270 bp,两者相差9个连续碱基,其翻译产物相差3个连续氨基酸残基;表达的融合蛋白以不溶性包涵体形式存在,纯化后得到纯度为97%的目的蛋白。结论:获得hMAM cDNA并发现一个新的hMAM突变体;融合蛋白的表达及纯化成功。
Objective ： To clone the cDNA in full-length of human mammaglobin, do prokaryotic expression and purify hMAM protein, as a basis for early diagnosis of breast cancer. Methods ： hMAM cDNA was amplified through RT-PCR from breast cancer tissue and breast cancer cell line MD-MB453, and the recombination pQE40-hMAM vector was constructed and expressed in E. Coli. M15 after induction by IPTG. The fusion protein was purified with Ni-NTA-His mffinity chromatography. Results： Two subtypes of hMAM cDNA and the hMAM （Isoform） cDNA were found, in which consisted of 270 bp, different from the wildtype hMAM cDNA of 279 bp on nine continuous base pair missing . The fusion protein formed inclusion body in prokaryotic expression system and the renatured protein was purified which purity was about 97%. Conclusion ： The recombinant hMAM was successfully purified.
出处
《医学研究生学报》
CAS
2006年第2期107-110,共4页
Journal of Medical Postgraduates
基金
重庆市科委应用基础研究基金资助项目(批准号:200407)
第三军医大学归国人员启动资金资助项目(批准号:XG200363)
关键词
乳腺癌
人乳腺珠蛋白
原核表达系统
蛋白纯化
Breast cancer
Human mammaglobin
Prokaryotic expression system
Protein purification
