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

  更多相關(guān)文章： 懸浮馴化 Marc-145-XF細(xì)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(guó)家,給世界養(yǎng)豬業(yè)造成嚴(yán)重經(jīng)濟(jì)損失,接種疫苗仍是最直接有效的預(yù)防措施。國(guó)內(nèi)外PRRS疫苗采用轉(zhuǎn)瓶貼壁培養(yǎng)Marc-145增殖病毒的生產(chǎn)工藝。生物反應(yīng)器懸浮培養(yǎng)細(xì)胞生產(chǎn)疫苗是世界范圍內(nèi)生物醫(yī)藥產(chǎn)業(yè)轉(zhuǎn)型升級(jí)的必由之路,而懸浮細(xì)胞系的建立是這一工藝的關(guān)鍵技術(shù)環(huán)節(jié)之一。本文采用驟減血清法與適應(yīng)降血清法,利用硅化搖瓶-搖床體系對(duì)低血清Marc-145貼壁細(xì)胞連續(xù)懸浮馴化傳代培養(yǎng)22代獲得了一株懸浮細(xì)胞,并對(duì)其在5L生物反應(yīng)器中大規(guī)模培養(yǎng)特性和病毒增殖能力進(jìn)行了研究。獲得以下主要研究結(jié)果:1.從CCTCC引進(jìn)一株貼壁培養(yǎng)Marc-145細(xì)胞,擴(kuò)增培養(yǎng)建立了細(xì)胞庫(kù),凍存40支,并對(duì)其生物學(xué)特性進(jìn)行了評(píng)價(jià)。引進(jìn)的Marc-145貼壁細(xì)胞的無(wú)菌檢查、支原體檢查、外源因子檢測(cè)結(jié)果均為陰性。復(fù)蘇的Marc-145貼壁細(xì)胞生長(zhǎng)穩(wěn)定,生長(zhǎng)曲線趨近于細(xì)胞的典型生長(zhǎng)曲線且基本呈"S"型,平均活力達(dá)到97%,最大增殖密度為57.8×104cells/mL,倍增時(shí)間為30.2 h。2.采取驟減血清法與適應(yīng)降血清法將Marc-145貼壁細(xì)胞的血清含量從10%降至2%,獲得一株2%低血清Marc-145貼壁細(xì)胞。該株低血清Marc-145貼壁細(xì)胞以1:3傳代,細(xì)胞生長(zhǎng)穩(wěn)定,形態(tài)良好,細(xì)胞最大增殖密度達(dá)4.03×105cells/mL,細(xì)胞活力98.5%,群體倍增時(shí)間為51.9 h。3.利用硅化搖瓶-搖床體系低血清Marc-145貼壁細(xì)胞連續(xù)懸浮馴化傳代培養(yǎng)22代得到一株Marc-145懸浮細(xì)胞,命名為Marc-145-XF。對(duì)Marc-145-XF細(xì)胞擴(kuò)增、建立細(xì)胞庫(kù)。馴化后的Marc-145-XF細(xì)胞無(wú)菌試驗(yàn)、支原體檢查、外源因子檢測(cè)結(jié)果均為陰性;染色體檢查與貼壁型Marc-145細(xì)胞一致。連續(xù)傳8代,細(xì)胞生長(zhǎng)正常,形態(tài)均一。傳代培養(yǎng)Marc-145-XF細(xì)胞以0.5×106cells/mL在無(wú)血清培養(yǎng)基中添加2%新生牛血清在培養(yǎng)至48 h進(jìn)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(shí)間27 h。4.接種豬藍(lán)耳病毒測(cè)得Marc-145-XF細(xì)胞的毒價(jià)為T(mén)CID50=10-4.74/0.1ml。5.利用5 L生物反應(yīng)器大規(guī)模培養(yǎng)Marc-145細(xì)胞時(shí),培養(yǎng)溫度為36.5℃,轉(zhuǎn)速設(shè)置為70 r/min,pH維持在6.5-7.15,溶氧設(shè)置在40%;細(xì)胞培養(yǎng)至96 h達(dá)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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