Xu Teng-fei, Zhang Wen-qing, Yu Hong, Li Dan
Pathogenic Biology Laboratory, Medical College, Qingdao University, Qingdao, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Mar;25(3):226-8.
To construct an eukaryotic vector encoding extracellular domain of human epidermal growth factor receptors (HER2), pcDNA6/v5-his-HER2, and to screen HER2 positive clones from mouse breast cancer cell line EMT6.
The extracellular domain of HER2 was amplified from pcDNA3.1-HER2 by PCR. pcDNA6/v5-his-HER2 was prepared by inserting the fragment into the plasmid pcDNA6/v5-his. Then the recombinant vector was identified by restriction enzyme and sequencing. Next, pcDNA6/v5-his-HER2 was transfected into the EMT6 cell line and the positive clones (EMT6/HER2) were screened with blasticidin. Finally, the expression of HER2 in EMT6/HER2 was detected by RT-PCR and immunohistochemistry.
The fragment of HER2 was amplified and pcDNA6/v5-his-HER2 was prepared successfully. No errors were found both in the sequence and ORF of the acquired fragment. The expected fragment of HER2 (1896 bp) was amplified from EMT6/HER2 by RT-PCR and positive signals of HER2 were detected in EMT6/HER2 by immunohistochemistry.
An eukaryotic plasmid encoding HER2 (pcDNA6/v5-his-HER2) has been constructed and a cell line expressing HER2 stably has been prepared successfully.
构建编码人表皮生长因子受体（HER2）胞外区的真核载体pcDNA6/v5-his-HER2，并从小鼠乳腺癌细胞系EMT6中筛选HER2阳性克隆。
通过PCR从pcDNA3.1-HER2中扩增HER2的胞外区。将该片段插入质粒pcDNA6/v5-his中制备pcDNA6/v5-his-HER2。然后通过限制性内切酶和测序鉴定重组载体。接下来，将pcDNA6/v5-his-HER2转染到EMT6细胞系中，并用杀稻瘟菌素筛选阳性克隆（EMT6/HER2）。最后，通过RT-PCR和免疫组织化学检测EMT6/HER2中HER2的表达。
成功扩增出HER2片段并制备了pcDNA6/v5-his-HER2。获得的片段在序列和开放阅读框中均未发现错误。通过RT-PCR从EMT6/HER2中扩增出预期的HER2片段（1896 bp），并通过免疫组织化学在EMT6/HER2中检测到HER2的阳性信号。
已构建编码HER2的真核质粒（pcDNA6/v5-his-HER2），并成功制备了稳定表达HER2的细胞系。
