本文選題：乙型腦炎病毒P_3株　切入點：Vero細(xì)胞　出處：《大連醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的： 為了提高產(chǎn)品的質(zhì)量,制造出更經(jīng)濟(jì)、更安全、更有效乙型腦炎滅活疫苗,我們從生產(chǎn)和檢定兩個方面著手：一是簡化生產(chǎn)程序,優(yōu)化制備疫苗過程中的各個環(huán)節(jié),選擇最簡單、最便宜又高效的方式方法進(jìn)行生產(chǎn),使乙腦疫苗更加安全、有效,且價格更便宜；二是完善檢定技術(shù),尋找最合適檢測方法,減少干擾因素,使檢測方法簡單,條件易于控制。制定嚴(yán)格的操作規(guī)程,使檢定結(jié)果最接近真實值,更能體現(xiàn)疫苗的真實情況,制造出更安全的人用乙型腦炎疫苗。 方法： 1.復(fù)蘇Vero細(xì)胞,并在細(xì)胞瓶中傳代、擴增,建立主細(xì)胞庫和工作細(xì)胞庫。 2.將乙腦病毒鼠腦P3株感染Vero細(xì)胞,制備乙型腦炎病毒Vero細(xì)胞P3株(P3MV株),建立主種子批和工作種子批。 3.用乙型腦炎病毒P3MV株接種Vero細(xì)胞,在適宜溫度下培養(yǎng),當(dāng)細(xì)胞病變達(dá)到+++-++++時,收獲病毒液。 4.將收獲的病毒液合并、滅活、濃縮、純化,經(jīng)稀釋制備成乙腦疫苗成品,再分裝、凍干 5.按照《中國藥典三部》2005版的要求,對制備疫苗的各個環(huán)節(jié)及半成品、成品進(jìn)行檢定。 結(jié)果： 1.擴增細(xì)胞,經(jīng)過傳代擴增,將Vero細(xì)胞131代定為主細(xì)胞庫,135代為工作細(xì)胞庫； 2.制備毒種庫,乙型腦炎病毒Vero細(xì)胞P3株,經(jīng)檢測毒力穩(wěn)定,免疫原性好,對現(xiàn)在流行的病毒株都具有良好的保護(hù)作用,建立病毒庫,將5代設(shè)為主種子批,8代設(shè)為工作種子批； 3.收獲病毒液,在接種病毒后每日觀察細(xì)胞病變,當(dāng)細(xì)胞病變達(dá)到+++-++++時收獲病毒液,每日收獲1次,連續(xù)收獲3-5天,合并后加入甲醛滅活。 4.將滅活實驗合格的病毒收獲液超濾濃縮50倍。 5.采用凝膠層析的方法純化疫苗,去除雜蛋白,降低不良反應(yīng)的發(fā)生。 6.將純化后原液稀釋分裝,制備成凍干或者水針。 7.按照藥典的要求對成品進(jìn)行全面檢定。 結(jié)論： 1.使用Vero細(xì)胞作為乙腦疫苗的生產(chǎn)基質(zhì),細(xì)胞學(xué)穩(wěn)定,沒有外源因子污染和致瘤性的危險,且培養(yǎng)簡單、使用代次高。 2.選用滅活工藝生產(chǎn)疫苗。采用經(jīng)典的甲醛作為滅活劑滅活乙腦病毒,按照1:2000的濃度加入甲醛,在4℃作用28天,通過細(xì)胞培養(yǎng)-動物法進(jìn)行滅活驗證。 3.采用Sepharose6FF凝膠過濾層析純化疫苗,其分離條件溫和、重現(xiàn)性好,還具有較高的回收率。 4.采用細(xì)胞蝕斑法代替小鼠法進(jìn)行病毒滴度檢測,試驗周期短、操作簡單、成本低。
[Abstract]:Objective:In order to improve the quality of the products and to produce a more economical, safer and more effective type B encephalitis inactivated vaccine, we should start from two aspects of production and verification: first, simplify the production process and optimize the various links in the preparation of the vaccine.Choose the simplest, cheapest and most efficient way to make the je vaccine safer, more effective, and cheaper; second, perfect the verification technology, look for the most appropriate detection method, reduce interference factors, and make the detection method simple.Conditions are easy to control.Make strict operation regulations, make the verification results closest to the true value, more can reflect the true situation of the vaccine, and make a safer vaccine for people with Japanese encephalitis.Methods:1.Vero cells were resuscitated, subcultured in cell flask, amplified, and the main cell bank and working cell bank were established.2.Vero cells were infected with Japanese encephalitis virus (je) P3 strain, and the main seed batch and working seed batch were established by preparing P3 strain of Japanese encephalitis virus (Vero) strain P3MV.3.Vero cells were inoculated with Japanese encephalitis virus (P3MV) strain and cultured at suitable temperature.4.Combine, inactivate, concentrate and purify the harvested viral solution, then dilute and prepare the finished product of je vaccine.5.According to the requirements of 2005 edition of Chinese Pharmacopoeia, the preparation of vaccine, semi-finished products and finished products were verified.Results:1.After subculture, 131-generation Vero cells were selected as working cell banks.2.The virus seed bank and P3 strain of Japanese encephalitis virus (Vero) cell were prepared. The virulence and immunogenicity of P3 strain of Japanese encephalitis virus were stable and immunogenicity was good. The virus bank was established and 8 generations of primary seed batches were set up as working seed batches.3.After inoculating the virus, the cytopathic effect was observed daily. When the cytopathic effect reached -, the virus solution was harvested once a day for 3-5 days, then added formaldehyde inactivated.4.Concentration 50 times of the viral harvest solution qualified for the inactivation test.5.The vaccine was purified by gel chromatography to remove the impurity protein and reduce the occurrence of adverse reactions.6.The purified solution was diluted and divided into freeze-dried or water needle.7.According to the requirements of the Pharmacopoeia, the finished product is fully verified.Conclusion:1.Using Vero cells as the production matrix of Japanese encephalitis vaccine, the cytology is stable, there is no risk of exogenous factor contamination and tumorigenicity, and the culture is simple, and the use of the vaccine is high.2.Use inactivation process to produce vaccine.The classical formaldehyde was used as the inactivated agent to inactivate Japanese encephalitis virus. Formaldehyde was added in the concentration of 1: 2000 and exposed at 4 鈩，

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