Marc-145悬浮培养细胞系的建立及培养研究

发布时间：2018-01-17 22:05

  本文关键词：Marc-145悬浮培养细胞系的建立及培养研究　出处：《西北民族大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 悬浮驯化 Marc-145-XF细胞 培养研究

【摘要】：猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS),是一种引起母猪繁殖障碍和新生仔猪呼吸道症状的病毒性传染病,具有很高的传染性和死亡率,几乎遍及世界所有养猪国家,给世界养猪业造成严重经济损失,接种疫苗仍是最直接有效的预防措施。国内外PRRS疫苗采用转瓶贴壁培养Marc-145增殖病毒的生产工艺。生物反应器悬浮培养细胞生产疫苗是世界范围内生物医药产业转型升级的必由之路,而悬浮细胞系的建立是这一工艺的关键技术环节之一。本文采用骤减血清法与适应降血清法,利用硅化摇瓶-摇床体系对低血清Marc-145贴壁细胞连续悬浮驯化传代培养22代获得了一株悬浮细胞,并对其在5L生物反应器中大规模培养特性和病毒增殖能力进行了研究。获得以下主要研究结果:1.从CCTCC引进一株贴壁培养Marc-145细胞,扩增培养建立了细胞库,冻存40支,并对其生物学特性进行了评价。引进的Marc-145贴壁细胞的无菌检查、支原体检查、外源因子检测结果均为阴性。复苏的Marc-145贴壁细胞生长稳定,生长曲线趋近于细胞的典型生长曲线且基本呈"S"型,平均活力达到97%,最大增殖密度为57.8×104cells/mL,倍增时间为30.2 h。2.采取骤减血清法与适应降血清法将Marc-145贴壁细胞的血清含量从10%降至2%,获得一株2%低血清Marc-145贴壁细胞。该株低血清Marc-145贴壁细胞以1:3传代,细胞生长稳定,形态良好,细胞最大增殖密度达4.03×105cells/mL,细胞活力98.5%,群体倍增时间为51.9 h。3.利用硅化摇瓶-摇床体系低血清Marc-145贴壁细胞连续悬浮驯化传代培养22代得到一株Marc-145悬浮细胞,命名为Marc-145-XF。对Marc-145-XF细胞扩增、建立细胞库。驯化后的Marc-145-XF细胞无菌试验、支原体检查、外源因子检测结果均为阴性;染色体检查与贴壁型Marc-145细胞一致。连续传8代,细胞生长正常,形态均一。传代培养Marc-145-XF细胞以0.5×106cells/mL在无血清培养基中添加2%新生牛血清在培养至48 h进行分种传代培养效果最佳,最大增殖密度为3.22×106cells/mL,倍增时间27 h。4.接种猪蓝耳病毒测得Marc-145-XF细胞的毒价为TCID50=10-4.74/0.1ml。5.利用5 L生物反应器大规模培养Marc-145细胞时,培养温度为36.5℃,转速设置为70 r/min,pH维持在6.5-7.15,溶氧设置在40%;细胞培养至96 h达到最大增殖密度2.85×106cells/mL。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs). It is a viral infectious disease that causes reproductive disorders in sows and respiratory symptoms of newborn piglets. It has a high infectious and mortality rate and is found in almost all pig breeding countries in the world, causing serious economic losses to the world pig industry. Vaccination is still the most direct and effective preventive measure. The production process of PRRS vaccine is transferred to bottle and wall to culture Marc-145 proliferative virus. Bioreactor suspension culture cell production vaccine is the world. The only way to the transformation and upgrading of biomedical industry within the scope. The establishment of suspension cell line is one of the key technologies of this process. A suspension cell was obtained by continuous suspension acclimation and subculture of low serum Marc-145 adherent cells in a silicified shaker shaker system for 22 generations. The characteristics of large scale culture and the ability of virus proliferation in 5L bioreactor were studied. The following main results were obtained: 1. A strain of Marc-145 cells was introduced from CCTCC. The cell bank was established, 40 of them were frozen, and their biological characteristics were evaluated. The introduced Marc-145 adherent cells were examined for asepsis and mycoplasma. The growth curve of Marc-145 adherent cells was stable, and the growth curve was similar to the typical growth curve of the cells. The average activity of the cells was 97%. The maximum density of proliferation was 57.8 脳 104 cells / mL. The doubling time was 30.2h.2.The serum content of Marc-145 adherent cells was reduced from 10% to 2% by the method of sudden reduction of serum and adaptive reduction of serum. A strain of 2% low serum Marc-145 adherent cells was obtained. The adherent cells of low serum Marc-145 were subcultured at 1: 3. The cells grew stably and had good morphology. The maximum cell proliferation density was 4.03 脳 105 cells / mL, and the cell viability was 98.5%. Population doubling time is 51.9. A strain of Marc-145 suspension cells was obtained by continuous suspension acclimation and subculture of low serum Marc-145 adherent cells in shaking flask and shaking bed system. It was named Marc-145-XF. the Marc-145-XF cells were amplified and the cell bank was established. The acclimated Marc-145-XF cells were tested for asepsis and mycoplasma. The results of exogenous factors were negative. Chromosome examination was consistent with adherent Marc-145 cells. Marc-145-XF cells were cultured with 0.5 脳 106 cells / mL in serum-free medium supplemented with 2% newborn bovine serum. H was the best for seed subculture. The maximum density of proliferation was 3.22 脳 106 cells / mL. The doubling time was 27 h. 4. The virulence of Marc-145-XF cells was determined to be 10 ~ (-4.74) / 0.1 ml. 5 by inoculating porcine blue ear disease virus. Marc-145 cells were cultured in L bioreactor on a large scale. The culture temperature was 36.5 鈩，

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