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t-PA突变体工程细胞株FSGGI48形态与其亲代细胞CHO-dhfr相似呈多角形,类似上皮细胞。在MTX加压至5×10~(-6)mol/L时,少数细胞形态略变瘦长,但仍呈多角形,因此该工程细胞株形态正常。抗体中和抑制试验及纤维蛋白板80℃加热试验显示,FSGGI48细胞表达产物与st-PA的活性均能被抗t-PA抗体所抑制,而胰酶的活性则不被抑制,且二者在80℃加热的纤维蛋白板上均不产生溶解圈,胰酶则产生溶解圈,由此可知,该细胞株表达产物为特异的rt-PA产品。细胞无血清培养上清经FA-PA检测,亚克隆株表达水平为4000IU/10~6细胞/24h。分别测定冻存3个月后复苏和体外传代3个月以上细胞的表达水平,结果显示部分亚克隆细胞株表达水平下降,多数仍为3000-4000IU/10~6细胞/24h,说明细胞株稳定性好。裸鼠试验表明,该工程株活细胞、细胞DNA、纯化的细胞表达产物均无致瘤性。支原体检查结果为阴性。染色体分析显示,FSGGI48细胞与其亲代细胞(CHO-dhfr细胞)染色体数目相同均为20条,但均有不同程度不同类型的畸变。CHO-dhfr细胞畸变率为6%。该工程细胞的畸变率为15%-18%,在允许范围内。结果证明,t-PA突变体细胞株FSGGI48为性能优良的工程细胞株。
The morphology of the t-PA mutant cell line FSGGI48 resembles that of its parental CHO-dhfr polyhedra, similar to epithelial cells. When MTX was pressurized to 5 × 10 ~ (-6) mol / L, the morphology of a few cells became thin and long, but still polygonal. Therefore, the morphology of this cell line was normal. Antibody neutralization inhibition test and heating test of fibrin plate at 80 ℃ showed that the activity of FSGGI48 cells and st-PA were inhibited by anti-t-PA antibody, while the activity of trypsin was not inhibited, 80 ℃ heated fibrin plate does not produce dissolved ring, trypsin is dissolved circle, which shows that the cell line expression products for the specific rt-PA products. The serum-free culture supernatant of the cells was detected by FA-PA, and the expression level of the subclone was 4000 IU / 10 ~ 6 cells / 24h. The results showed that the expression levels of some subclone cell lines were decreased, most of them remained at 3000-4000IU / 10 ~ 6 cells / 24h, which indicated that the cell lines were stable Good sex. Nude mice test showed that the engineering strain live cell, cell DNA, purified cell expression products are not tumorigenic. Mycoplasma test results were negative. Chromosome analysis showed that there were 20 chromosomes in FSGGI48 cells and their parental cells (CHO-dhfr cells), but all of them had different types of aberrations. CHO-dhfr cell aberration rate was 6%. The engineering cell aberration rate of 15% -18%, within the allowable range. The results demonstrate that the t-PA mutant strain FSGGI48 is an engineered cell line with superior performance.