本文關鍵詞：Marc-145懸浮培養(yǎng)細胞系的建立及培養(yǎng)研究　出處：《西北民族大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 懸浮馴化 Marc-145-XF細胞 培養(yǎng)研究

【摘要】：豬繁殖與呼吸綜合征(porcine reproductive and respiratory syndrome,PRRS),是一種引起母豬繁殖障礙和新生仔豬呼吸道癥狀的病毒性傳染病,具有很高的傳染性和死亡率,幾乎遍及世界所有養(yǎng)豬國家,給世界養(yǎng)豬業(yè)造成嚴重經(jīng)濟損失,接種疫苗仍是最直接有效的預防措施。國內(nèi)外PRRS疫苗采用轉瓶貼壁培養(yǎng)Marc-145增殖病毒的生產(chǎn)工藝。生物反應器懸浮培養(yǎng)細胞生產(chǎn)疫苗是世界范圍內(nèi)生物醫(yī)藥產(chǎn)業(yè)轉型升級的必由之路,而懸浮細胞系的建立是這一工藝的關鍵技術環(huán)節(jié)之一。本文采用驟減血清法與適應降血清法,利用硅化搖瓶-搖床體系對低血清Marc-145貼壁細胞連續(xù)懸浮馴化傳代培養(yǎng)22代獲得了一株懸浮細胞,并對其在5L生物反應器中大規(guī)模培養(yǎng)特性和病毒增殖能力進行了研究。獲得以下主要研究結果:1.從CCTCC引進一株貼壁培養(yǎng)Marc-145細胞,擴增培養(yǎng)建立了細胞庫,凍存40支,并對其生物學特性進行了評價。引進的Marc-145貼壁細胞的無菌檢查、支原體檢查、外源因子檢測結果均為陰性。復蘇的Marc-145貼壁細胞生長穩(wěn)定,生長曲線趨近于細胞的典型生長曲線且基本呈"S"型,平均活力達到97%,最大增殖密度為57.8×104cells/mL,倍增時間為30.2 h。2.采取驟減血清法與適應降血清法將Marc-145貼壁細胞的血清含量從10%降至2%,獲得一株2%低血清Marc-145貼壁細胞。該株低血清Marc-145貼壁細胞以1:3傳代,細胞生長穩(wěn)定,形態(tài)良好,細胞最大增殖密度達4.03×105cells/mL,細胞活力98.5%,群體倍增時間為51.9 h。3.利用硅化搖瓶-搖床體系低血清Marc-145貼壁細胞連續(xù)懸浮馴化傳代培養(yǎng)22代得到一株Marc-145懸浮細胞,命名為Marc-145-XF。對Marc-145-XF細胞擴增、建立細胞庫。馴化后的Marc-145-XF細胞無菌試驗、支原體檢查、外源因子檢測結果均為陰性;染色體檢查與貼壁型Marc-145細胞一致。連續(xù)傳8代,細胞生長正常,形態(tài)均一。傳代培養(yǎng)Marc-145-XF細胞以0.5×106cells/mL在無血清培養(yǎng)基中添加2%新生牛血清在培養(yǎng)至48 h進行分種傳代培養(yǎng)效果最佳,最大增殖密度為3.22×106cells/mL,倍增時間27 h。4.接種豬藍耳病毒測得Marc-145-XF細胞的毒價為TCID50=10-4.74/0.1ml。5.利用5 L生物反應器大規(guī)模培養(yǎng)Marc-145細胞時,培養(yǎng)溫度為36.5℃,轉速設置為70 r/min,pH維持在6.5-7.15,溶氧設置在40%;細胞培養(yǎng)至96 h達到最大增殖密度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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