流感病毒裂解疫苗工藝研究
發(fā)布時(shí)間：2018-07-14 12:10
【摘要】： 流感病毒具有高度的傳染性,能引起急性呼吸道傳染疾病,流行特點(diǎn)是在短時(shí)間內(nèi)突然爆發(fā),迅速蔓延,造成不同程度的流行,包括世界大流行、局部流行暴發(fā)及擴(kuò)散。其發(fā)病率極高,同時(shí)伴有很高的死亡率。目前尚無(wú)特效藥,接種流感疫苗是被公認(rèn)的預(yù)防流感發(fā)生與傳播的最佳方法,用雞胚作為病毒生長(zhǎng)介質(zhì)也是目前流感疫苗生產(chǎn)最常用的培養(yǎng)基質(zhì)。 我們首先將來(lái)源于WHO流感參考和研究合作中心的H1N1、H3N2、B三種不同型別的流感病毒毒株分別接種于SPF級(jí)雞胚,經(jīng)培養(yǎng)后,收獲其尿囊液,制備主代和工作種子批。將工作種子批接種來(lái)源于封閉式房舍內(nèi)飼養(yǎng)的健康雞群雞胚進(jìn)行大規(guī)模培養(yǎng),收獲病毒液,然后經(jīng)超濾濃縮、純化、裂解、滅活、二次純化等步驟,獲得三個(gè)型別的單價(jià)病毒液。在這過(guò)程中首先明確了適宜的毒種稀釋倍數(shù)和病毒收獲時(shí)間,然后摸索了蔗糖密度梯度離心純化應(yīng)該收集的病毒峰,并對(duì)工藝中裂解劑以及滅活劑的選用進(jìn)行了研究,確定了生產(chǎn)工藝。 選用兩種裂解劑Triton X100和Triton N101,以不同濃度對(duì)流感病毒單價(jià)純化液作用,通過(guò)電鏡進(jìn)行裂解效果觀察,確定最佳裂解時(shí)間,用單向免疫擴(kuò)散法檢測(cè)HA含量作為評(píng)價(jià)指標(biāo)。結(jié)果表明:Triton X100的裂解效果較優(yōu)越,0.5%～1%,Triton X100作用90min可使流感病毒裂解。我們?cè)诒敬窝芯恐?將二乙烯亞胺(Binary Ethylene Imine,簡(jiǎn)稱BEI)引入制備流感病毒滅活疫苗當(dāng)中,與甲醛做對(duì)照,BEI的最佳滅活時(shí)間在24～36h之間,甲醛的為3h;BEI的滅活后的免疫效果明顯優(yōu)于甲醛。運(yùn)用RT-PCR的方法測(cè)定出病毒8個(gè)基因滅活前后的變化,表明BEI滅活病毒的同時(shí)將8個(gè)基因序列完全破壞,而甲醛破壞不完全,從分子生物學(xué)水平證明經(jīng)BEI滅活的流感滅活疫苗更加安全、可靠。通過(guò)單向免疫擴(kuò)散試驗(yàn)對(duì)兩種滅活劑的滅活效果做進(jìn)一步驗(yàn)證,發(fā)現(xiàn)雖然BEI滅活病毒的時(shí)間長(zhǎng),但是其樣品中HA含量較甲醛滅活的高,但BEI能否用于人用疫苗的制備還需進(jìn)一步研究進(jìn)行大量的臨床實(shí)驗(yàn)。 本研究對(duì)使用雞胚培養(yǎng)流感病毒制備流感裂解疫苗生產(chǎn)工藝的幾個(gè)重要環(huán)節(jié)進(jìn)行試驗(yàn)摸索,為將來(lái)生產(chǎn)工藝的進(jìn)一步改進(jìn)提供了研究基礎(chǔ)。
[Abstract]:Influenza virus is highly infectious and can cause acute respiratory tract transmission disease. The epidemic characteristic is a sudden outbreak in a short period of time, rapid spread, resulting in different levels of epidemic, including the world pandemic, local outbreak and spread. Its incidence is extremely high, accompanied by a high mortality rate. At present, there is no specific drug, inoculation of influenza vaccine is recognized as the best way to prevent the occurrence and transmission of influenza, chicken embryo as the medium of virus growth is also the most commonly used culture substrate for influenza vaccine production at present. We first inoculated three different types of influenza virus strains from WHO Influenza reference and Research collaborating Center into SPF chicken embryos. After culturing, we harvested their allantoic fluid and prepared the main generation and working seed batch. Batch inoculation of working seeds from healthy chicken embryos raised in closed houses was carried out on a large scale, and virus solution was harvested, then concentrated by ultrafiltration, purified, decomposed, inactivated, re-purified and so on. Three types of monovalent virus solution were obtained. In the process, the suitable dilution times and harvest time of virus were determined, then the virus peak should be collected by sucrose density gradient centrifugation was explored, and the selection of cracking agent and inactivating agent in the process was studied. The production process was determined. Two kinds of cracking agents Triton X100 and Triton N101 were used to treat influenza virus monovalent purified solution with different concentrations. The best lysis time was determined by electron microscope, and the HA content was detected by one-way immunodiffusion. The results showed that the cleavage effect of Triton X100 was better than that of Triton X100. Triton X100 could make influenza virus lyse with Triton X100. In this study, we introduced binary Ethylene Imine (BEI) into the preparation of Influenza virus inactivated vaccine. The best inactivation time of BEI compared with formaldehyde was between 24 and 36 hours. The immune effect of the inactivated 3 h BEI of formaldehyde was obviously better than that of formaldehyde. The changes before and after the inactivation of the 8 genes of the virus were determined by RT-PCR. The results showed that the BEI inactivated virus completely destroyed the sequence of the 8 genes while the formaldehyde was not completely destroyed. It is proved that the inactivated influenza vaccine inactivated by BEI is more safe and reliable from the level of molecular biology. The inactivation effect of two inactivated agents was further verified by one-way immunodiffusion test. It was found that although BEI inactivated the virus for a long time, the HA content in the sample was higher than that in formaldehyde inactivation. However, whether BEI can be used in the preparation of human vaccine needs further research and a large number of clinical trials. In this study, several important links in the production process of influenza lytic vaccine using chicken embryo culture influenza virus were tested and explored, which provided the basis for further improvement of the production process in the future.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
本文編號(hào)：2121602
[Abstract]:Influenza virus is highly infectious and can cause acute respiratory tract transmission disease. The epidemic characteristic is a sudden outbreak in a short period of time, rapid spread, resulting in different levels of epidemic, including the world pandemic, local outbreak and spread. Its incidence is extremely high, accompanied by a high mortality rate. At present, there is no specific drug, inoculation of influenza vaccine is recognized as the best way to prevent the occurrence and transmission of influenza, chicken embryo as the medium of virus growth is also the most commonly used culture substrate for influenza vaccine production at present. We first inoculated three different types of influenza virus strains from WHO Influenza reference and Research collaborating Center into SPF chicken embryos. After culturing, we harvested their allantoic fluid and prepared the main generation and working seed batch. Batch inoculation of working seeds from healthy chicken embryos raised in closed houses was carried out on a large scale, and virus solution was harvested, then concentrated by ultrafiltration, purified, decomposed, inactivated, re-purified and so on. Three types of monovalent virus solution were obtained. In the process, the suitable dilution times and harvest time of virus were determined, then the virus peak should be collected by sucrose density gradient centrifugation was explored, and the selection of cracking agent and inactivating agent in the process was studied. The production process was determined. Two kinds of cracking agents Triton X100 and Triton N101 were used to treat influenza virus monovalent purified solution with different concentrations. The best lysis time was determined by electron microscope, and the HA content was detected by one-way immunodiffusion. The results showed that the cleavage effect of Triton X100 was better than that of Triton X100. Triton X100 could make influenza virus lyse with Triton X100. In this study, we introduced binary Ethylene Imine (BEI) into the preparation of Influenza virus inactivated vaccine. The best inactivation time of BEI compared with formaldehyde was between 24 and 36 hours. The immune effect of the inactivated 3 h BEI of formaldehyde was obviously better than that of formaldehyde. The changes before and after the inactivation of the 8 genes of the virus were determined by RT-PCR. The results showed that the BEI inactivated virus completely destroyed the sequence of the 8 genes while the formaldehyde was not completely destroyed. It is proved that the inactivated influenza vaccine inactivated by BEI is more safe and reliable from the level of molecular biology. The inactivation effect of two inactivated agents was further verified by one-way immunodiffusion test. It was found that although BEI inactivated the virus for a long time, the HA content in the sample was higher than that in formaldehyde inactivation. However, whether BEI can be used in the preparation of human vaccine needs further research and a large number of clinical trials. In this study, several important links in the production process of influenza lytic vaccine using chicken embryo culture influenza virus were tested and explored, which provided the basis for further improvement of the production process in the future.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
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，本文編號(hào)：2121602
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