适于无血清贴壁培养的抗凋亡宿主细胞系CHO-IVB2的构建

应用无血清培养基培养CHO细胞时，由于没有血清提供各种贴壁因子，细胞以悬浮的方式生长。在实际的大规模细胞培养中，CHO细胞往往以贴壁方式培养，要么贴壁于悬浮的微载体中，要么贴壁于固定的聚酯盘状介质或中空纤维中，而很少直接悬浮于培养基中。在无血清培养基中，Vitronectin单一组分可以促使CHO细胞的贴壁和扩增。通过双表达lgf-1和Bcl-2基因，已经构建了可以在无蛋白培养基IMEM中抗凋亡生长的细胞株CHO-IB3。在此基础上，构建了可以同时表达Igf-1、Vitronectin和Bcl-2三个蛋白的三顺反子表达载体pCI—NII—IVB。将该载体转染于CHO—dhfr^-细胞中，构建了一个细胞株CHO—IVB2。该细胞株可以在无蛋白培养基中抗凋亡生长，适于以贴壁的方式大规模培养，用于大量生产外源目的蛋白。

Construction of CHO-IVB, A Serum-independent, Apoptosis-resistant Cell Line that can Grow in Adherence

Without serum to provide adherent factors, CHO-dhfr - cells grow in suspension when cultured in serum-free medium. Although this offers advantages in some applications, in most production systems adherent cell growth is preferable. Gene transfection, clonal selection and amplification can be easier for adherent cells; the density of immobilized cells is often higher than those in suspension culture, which results in a higher protein productivity; washout of cells by perfused medium during continuous fermentation can be avoided; for high-throughput microplate assays, adherent cells are preferred to facilitate medium changes and cell washing. It has been proved that purified vitronectin alone was able to mediate attachment and spreading of CHO cells in serum-free medium. So we constructed a tricistronic expression vector expressing Igf-1, Vitronectin and Bcl-2 at the same time. The vector was transfected into CHO-dhfr - cells and one clone, namely CHO-IVB2, expressing high level of the three proteins was screened out by Western blot. The cell line showed similar apoptosis-resistant and serum-independent properties to CHO-IB, an engineered cell line constructed before. When cultured in IMEM protein-free medium without any components supplemented, CHO-IVB can grow adherently. The viable cell numbers and growth rate of CHO-IVB were much higher than CHO-IB, making CHO-IVB an apoptosis-resistant host for production of recombinant proteins which can grow adherently in protein-free medium.