人凝血因子Ⅶ基因在CHO-K1细胞中的瞬时表达与鉴定

Transient expression and identification of human coagulation factor Ⅶ in CHO-K1 cell

目的在CHO-K1细胞中瞬时表达所克隆的人凝血因子Ⅶ(FⅦ)的cDNA序列并表达其产物。方法首先从HepG2细胞中克隆人FⅦcDNA序列,将克隆得到的序列连接到pMD18-T载体上经测序验证后,将其与pcDNA3.1载体连接,构建得到重组表达质粒pcDNA3.1-FⅦ。将构建得到的表达质粒瞬时转染CHO-K1细胞,72 h取样,采用ELISA、Western blotting等方法对细胞上清液和细胞裂解物作鉴定。结果克隆得到的序列经NCBI比对后为FⅦ转录突变体Ⅱ基因序列,克隆得到的基因未见氨基酸突变,重组表达质粒pcDNA3.1-FⅦ经PCR、双酶切、测序验证正确。ELISA法未在上清中检测到目的蛋白,而在细胞裂解液中检测到了目的蛋白;Western blotting检测到表达产物有与FⅦ标准品类似的分子量为50 kD左右的特异性条带。结论在转染后的CHO-K1细胞裂解液上清中成功表达了hFⅦ蛋白。

Objective To transient express the human coagulation factor Ⅶ gene in CHO-K1 cell. Methods The hu- man coagulation factor Ⅶ cDNA was amplified from total RNA of HepG2 cell line using RT-PCR. After identified by DNA se- quencing,the correct cDNA was cloned into pcDNA3.1 vector to get expression vector pcDNA3.1-FⅦ which was transfected into CHO-K1 cell. Culture supernatant and cell lysate were collected at 72 h after transfection and detected by Western blot- ting and ELISA. Results Sequence analysis showed that the fragment we obtained was the human coagulation factor Ⅶ tran- script variant Ⅱ gene, and further analysis demonstrated that no amino acid was changed in the sequence. The pcDNA3.1-FⅦ expression vector was constructed correctly by PCR and enzyme cut. ELISA showed that hFⅦ was detected in the cell lysate but not in the culture supernatant, and Western blotting further confirmed that the protein was approximately 50 kD. Conclu- sion Human coagulation factor VII was succefully expressed in CHO-K1 cell lysate.