乳腺癌相关的乳腺珠蛋白基因的克隆、表达及纯化

目的 克隆人乳腺珠蛋白(hMAM)基因,原核表达重组蛋白并纯化,为建立一种新的乳腺癌诊断方法奠定基础.方法 从人乳腺癌组织中提取总RNA,经RT-PCR合成cDNA,设计特异性的引物,用PCR的方法扩增出目的片段,构建PET-32A-hMAM和PGEX-4T1 -hMAM表达载体,在BL21菌中表达hMAM重组蛋白,纯化,并进行SDS-PAGE及Western blotting鉴定.结果 经RT-PCR、PCR扩增后得到一条207 bp的DNA片段,构建载体后DNA测序,结果与预期序列一致.表达和纯化的融合蛋白相对分子质量大约为28 000,与预期结果一致.结论 成功克隆hMAM基因,并获得高纯度的hMAM重组蛋白,为进一步开发hMAM诊断试剂打下基础.

Construction,expression and purification of hMAM fusion protein

Objective To express human mammaglobin（hMAM） fusion protein. Methods The gene encoding hMAM was cloned by RT-PCR from the total RNA of breast cancer tissues. Recombinant plasmids PET- 32a-hMAM and PGEX-4T1-hMAM were constructed and used to transfect E. coli BL21. hMAM was expressed, purified and identified by SDS-PAGE and Western blotting. Results The extracted DNA fragment about 207bp was confirmed as hMAM gene by DNA sequencing. The relative molecular mass of the expressed fusion protein in E. coli was about 28 000. Conclusions hMAM was expressed and purified successfully. The study lays a foundation for the further development of hMAM diagnostic reagent.