【摘要】：近年來我國發(fā)生了多次禽流感疫情,造成了巨大的社會恐慌和經(jīng)濟(jì)損失。疫苗接種是目前預(yù)防和控制流感最經(jīng)濟(jì)有效的措施。在我國獲得批準(zhǔn)使用的流感疫苗均以雞胚作為病毒生產(chǎn)基質(zhì)。鑒于雞胚的供應(yīng)需要較長的時間,不能在短期內(nèi)提供足夠的疫苗以應(yīng)對隨時可能爆發(fā)的流感大流行。而Vero細(xì)胞能夠利用細(xì)胞工廠和微載體-生物反應(yīng)器系統(tǒng)進(jìn)行大規(guī)模培養(yǎng),生產(chǎn)過程易于控制及實現(xiàn)規(guī)�；a(chǎn),且培養(yǎng)出的流感病毒較雞胚培養(yǎng)的病毒變異小,用其生產(chǎn)出的流感疫苗與當(dāng)時流行株匹配程度高。因此,開發(fā)Vero細(xì)胞大流行流感疫苗顯得尤為必要。在國內(nèi)外,細(xì)胞工廠培養(yǎng)技術(shù)已經(jīng)是非常成熟和普遍的體外大量培養(yǎng)細(xì)胞的技術(shù),如培養(yǎng)KMB-17、2BS、MRC-5等細(xì)胞。其具有便捷靈活安全的操作方式,較少的占用空間,可控性好,既有優(yōu)于轉(zhuǎn)瓶的優(yōu)勢,又沒有類似生物反應(yīng)器的應(yīng)用限制。本文主要考察了 Vero細(xì)胞在細(xì)胞工廠中的生長情況以及利用細(xì)胞工廠培養(yǎng)流感病毒條件的優(yōu)化,篩選出了細(xì)胞工廠培養(yǎng)H5N1流感病毒較適培養(yǎng)條件為:胞齡為60～72 h、TPCK-胰酶含量為2μg/ml,MOI為0.1,收毒時間為接種后72 h,培養(yǎng)溫度為33±0.5℃,維持液pH值約為7.6,病毒滴度達(dá)到1:256～1:1024。免疫熒光檢測發(fā)現(xiàn)H5N1亞型流感病毒能夠較好地感染Vero細(xì)胞;H5N1 Va流感病毒(A/Anhui/1/2005 va)在Vero細(xì)胞上擴(kuò)增的過程中型別保持不變;基因序列比對結(jié)果證實H5N1Va兩個表面抗原基因確實來源于A/Anhui/1/2005(H5N1)。由于Vero細(xì)胞為傳代細(xì)胞系,生產(chǎn)時存在一些問題,如殘余宿主蛋白可能引起過敏反應(yīng)以及宿主細(xì)胞DNA有致瘤性風(fēng)險。疫苗生產(chǎn)時需要控制Vero細(xì)胞的代次在150代以內(nèi),并進(jìn)行后處理純化以減少殘余Vero細(xì)胞DNA和蛋白含量。本文對Vero細(xì)胞流感疫苗后處理純化工藝進(jìn)行了摸索,確定使用0.65、0.45、0.22 μm濾芯進(jìn)行三級過濾澄清、300 KD超濾膜包濃縮、加Benzonase酶37 ℃處理病毒濃縮液4 h、1000 KD超濾膜包低于5 psi洗濾濃縮,然后再經(jīng)Sepharose 4FF和DEAE Sepharose FF兩步柱層析純化。病毒回收率70%以上,殘余DNA含量小于100 pg/劑,宿主蛋白含量小于2 μg/劑,BSA含量小于50 ng/劑,都達(dá)到了藥典要求。該研究為以Vero細(xì)胞為基質(zhì)的大流行流感疫苗的研發(fā)奠定了基礎(chǔ)。
[Abstract]:In recent years, there have been many outbreaks of avian flu in China, resulting in huge social panic and economic losses. Vaccination is the most economical and effective measure to prevent and control influenza. Chicken embryo is used as the production medium of influenza vaccine approved in China. Given the long supply of chicken embryos, it is not possible to provide enough vaccines in the short term for an influenza pandemic at any time. However, Vero cells can be cultured on a large scale by cell factory and microcarrier bioreactor system. The production process is easy to control and realize large-scale production, and the influenza virus produced is less variant than that of chicken embryo. The flu vaccine produced by the vaccine matched well with the prevailing strain at the time. Therefore, the development of Vero cell pandemic influenza vaccine is particularly necessary. At home and abroad, cell factory culture technology has been a very mature and universal in vitro culture of cells, such as the culture of KMB-17,2BS,MRC-5 cells. The utility model has the advantages of convenient, flexible and safe operation, less space occupation, good controllability, which is superior to the flask rotation, and has no application limitations similar to that of bioreactor. In this paper, the growth of Vero cells in cell factories and the optimization of the conditions for the cultivation of influenza viruses in cell factories were investigated. The optimum conditions for the culture of H5N1 influenza viruses in cell factories were as follows: the cell age was 6072h. The TPCK- trypsin content was 2 渭 g / ml moi 0.1, the inoculation time was 72 h, the culture temperature was 33 鹵0.5 鈩，

本文編號：2399212