本文選題：Vero細(xì)胞 + 冷適應(yīng)�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：流感病毒是嚴(yán)重威脅人類健康的一種急性呼吸道傳染病病原體,每年在全球引起較高的發(fā)病率和死亡率。接種疫苗是預(yù)防和控制流感流行的最有效手段。目前批準(zhǔn)使用的流感疫苗主要有三類：滅活苗、減毒活疫苗和重組HA疫苗。國(guó)內(nèi)使用的僅有滅活的裂解疫苗或亞單位疫苗。流感減毒活疫苗以類似自然感染途徑的噴鼻接種,既可刺激機(jī)體產(chǎn)生體液免疫,又能引起局部黏膜免疫和細(xì)胞免疫應(yīng)答,其免疫效果安全有效。流感減毒活疫苗的制備主要是基于流感病毒的基因組為分節(jié)段的RNA,可以利用遺傳重配技術(shù)將減毒供體株和流行株進(jìn)行重配,重配株獲得來(lái)自流行株的表面抗原基因和減毒株的表型特征。在該領(lǐng)域研究最多的是俄羅斯和美國(guó),美國(guó)采用俄羅斯提供的冷適應(yīng)供體株A/Ann Arbor/6/60(H2N2)和B/Ann Arbor/1/66制備的第一支三價(jià)流感減毒活疫苗Flumist(?)于2003年在美國(guó)上市。目前使用的流感減毒活疫苗均以雞胚作為培養(yǎng)基質(zhì)。以雞胚作為基質(zhì)生產(chǎn)的流感減毒活疫苗,存在外源因子污染和將雞傳染病病原體借由活疫苗接種播散到人群中的風(fēng)險(xiǎn)；同時(shí)雞胚供應(yīng)周期長(zhǎng),當(dāng)大流感來(lái)襲時(shí)難以滿足大規(guī)模生產(chǎn)需求,尤其是禽流感病毒來(lái)襲時(shí)雞胚來(lái)源更受影響。鑒于此,WHO鼓勵(lì)開(kāi)展以哺乳動(dòng)物細(xì)胞代替雞胚作為流感疫苗生產(chǎn)基質(zhì)的研究。Vero細(xì)胞是WHO批準(zhǔn)生產(chǎn)人用疫苗的傳代細(xì)胞系,但對(duì)流感病毒不敏感,產(chǎn)毒量不穩(wěn)定。目前未見(jiàn)以Vero細(xì)胞為基質(zhì)生產(chǎn)的季節(jié)性流感病毒減毒活疫苗,選育流感病毒Vero細(xì)胞冷適應(yīng)株對(duì)于流感減毒活疫苗的研發(fā)至關(guān)重要。目前世界各國(guó)選育出的Vero細(xì)胞冷適應(yīng)株極為稀少,僅見(jiàn)一株A/Singapore/1/57ca(H2N2)報(bào)道。本實(shí)驗(yàn)室經(jīng)過(guò)梯度降溫培養(yǎng)的方法選育出一株Vero細(xì)胞冷適應(yīng)流感病毒株A/Yunnan/1/2005Vca(H3N2),已經(jīng)在25℃連續(xù)傳72代,病毒的滴度穩(wěn)定在7.8-8.21gTCID5o/mL。經(jīng)鑒定A/Yunnan/1/2005Vca(H3N2)具有冷適應(yīng)表型(ca)、溫度敏感表型(ts)和減毒表型(att),并且該毒株在雪貂和小鼠模型上表現(xiàn)出較好的安全性和免疫原性,能有效刺激機(jī)體產(chǎn)生細(xì)胞免疫和體液免疫,并能保護(hù)免疫動(dòng)物抵抗同亞型流感病毒的致死性攻擊,提示A/Yunnan/1/2005Vca(H3N2)有可能是制備流感病毒Vero細(xì)胞減毒活疫苗株的理想候選供體株。在本研究中,以實(shí)驗(yàn)室選育的A/Yunnan/1/2005 Vca(H3N2)毒株與WHO提供的疫苗生產(chǎn)用流行株A/Solomom Islands/3/2006(H1N1)進(jìn)行傳統(tǒng)遺傳重配方法學(xué)的研究,以建立穩(wěn)定而快速的遺傳重配技術(shù),并對(duì)重配株病毒reA/SI/2006(H1N1)Vca進(jìn)行生物學(xué)表型特征的鑒定與疫苗安全性和免疫原性的研究,以證明供體株A/Yunnan/1/2005Vca(H3N2)能作為流感病毒Vero細(xì)胞減毒活疫苗的母本毒株。以兩種病毒同時(shí)感染Vero細(xì)胞、MDCK細(xì)胞和雞胚三種培養(yǎng)基質(zhì),加羊抗A/Yunnan/1/2005Vca(H3N2)病毒血清中和供體株,并在Vero細(xì)胞25℃條件下培養(yǎng)篩選,最終分別在Vero細(xì)胞33℃和MDCK細(xì)胞25℃成功重配獲得能在Vero細(xì)胞25℃生長(zhǎng)的毒株reA/SI/2006(H1N1)Vca。采用血凝抑制實(shí)驗(yàn)和單向免疫擴(kuò)散試驗(yàn)對(duì)重配毒進(jìn)行型別鑒定,結(jié)果證實(shí)重配株病毒抗原性與流行株A/Solomom slands/3/2006(H1N1)一致。進(jìn)一步的基因測(cè)序結(jié)果表明,重配株reA/SI/2006(H1N1)Vca表面抗原基因HA和NA來(lái)自流行株A/Solomom Islands/3/2006(H1N1),6個(gè)內(nèi)部基因來(lái)自供體株A/Yunnan/1/2005Vca(H3N2)。在經(jīng)過(guò)優(yōu)化的培養(yǎng)條件下,將重配毒株reA/SI/2006(H1N1)Vca在Vero細(xì)胞25℃連續(xù)傳18代,病毒滴度在7.51gTCID50/mL以上,傳代過(guò)程中病毒型別不發(fā)生改變。重配病毒reA/SI/2006(H1N1)Vca在25℃、33℃和39℃的感染性滴度分別為6.9、7.8和3.51gTCID50/mL,具有ca、ts表型。以106 TCID50的病毒量滴鼻接種BALB/c小鼠,免后連續(xù)7天測(cè)定小鼠鼻夾和肺組織的病毒載量,重配株病毒reA/SI/2006(H1N1)Vca組在前五天,上下呼吸道的病毒載量均比疫苗株低了至少31gTCID50/g,說(shuō)明重配病毒具有減毒表型。以105TCID50、106TCID50和107TCID50三種劑量對(duì)BALB/c小鼠進(jìn)行二次免疫,能有效刺激小鼠產(chǎn)生針對(duì)流行株病毒A/Solomom Islands/3/2006(H1N1)的血凝抑制抗體和中和抗體,并能促進(jìn)粘膜sIgA的分泌。接種重配病毒reA/SI/2006(H1N1)Vca后,不僅完全保護(hù)小鼠免受同亞型流感病毒A/JN/2009(H1N1) 50MLD50病毒量的致死性攻擊,而且能完全抑制攻擊病毒在小鼠體內(nèi)的復(fù)制。本研究表明,reA/SI/2006(H1N1)Vca是以遺傳重配技術(shù)獲得的既帶有流行株的表面抗原基因,又能在Vero細(xì)胞25℃生長(zhǎng),并具有減毒表型特征的重配病毒,因此A/Yunnan/1/2005Vca(H3N2)可以用作流感病毒Vero細(xì)胞冷適應(yīng)減毒活疫苗的母本株。
[Abstract]:Influenza virus is an acute respiratory infectious disease pathogen that seriously threatens human health. It causes high incidence and mortality in the world every year. Vaccination is the most effective means to prevent and control influenza epidemic. There are three main types of influenza vaccine approved at present: inactivated vaccine, live attenuated vaccine and recombinant HA vaccine. The only inactivated lysate vaccine or subunit vaccine is used. The vaccine can stimulate the body to produce humoral immunity and cause local mucosal immunity and cellular immune response. The immune effect is safe and effective. The preparation of the live attenuated influenza vaccine is mainly based on the gene of influenza virus. RNA, a segment of the segment, can be used to rematch the antivirus and epidemic strains by genetic matching. The redistribution plants obtain the surface antigen gene and the phenotypic characteristics of the antivirus strains from the epidemic strains. In this field, the most research is Russia and the United States, the United States adopts the cold adapted donor plant, A/Ann Arbor/6/60 (H2N2) and B/A, used by the United States. The first trivalent live attenuated influenza vaccine (Flumist), prepared by NN Arbor/1/66, was listed in the United States in 2003. At the same time, chicken embryo supply cycle is long, and when the pandemic attacks are difficult to meet mass production demand, especially when the avian influenza virus comes in, the chicken embryo is more affected. In view of this, WHO encourages the use of mammalian cells instead of chicken embryos as the substrate for influenza vaccine production..Vero cells are WHO approving producer vaccines. The cell line is not sensitive to influenza virus, and the yield of the virus is unstable. There is no seasonal influenza virus vaccine produced by Vero cells. It is very important to select cold adapted strains of influenza virus Vero cells for the research and development of the live attenuated influenza vaccine. At present, the cold adapted strains of Vero cells selected from all over the world are rare. Only one strain of A/Singapore/1/57ca (H2N2) was reported. In this laboratory, a Vero cell cold adapted influenza virus strain, A/Yunnan/1/2005Vca (H3N2), has been bred by gradient cooling culture, and has been passed on for 72 generations at 25 degrees centigrade. The titer of the virus is stable in the 7.8-8.21gTCID5o/mL. confirmed A/Yunnan/1/2005Vca (H3N2) with the cold adaptation phenotype (CA) and temperature. The degree sensitive phenotype (TS) and the attenuated phenotype (ATT), and the strain in ferrets and mouse models show good safety and immunogenicity, can effectively stimulate the body to produce cellular and humoral immunity, and protect the immune animals against the lethal attack of the same subtype influenza virus, suggesting that A/Yunnan/1/2005Vca (H3N2) may be prepared. An ideal candidate donor strain of a live attenuated influenza virus Vero cell vaccine strain. In this study, a laboratory selected A/Yunnan/1/2005 Vca (H3N2) strain and a popular strain A/Solomom Islands/3/2006 (H1N1) provided by WHO were used to study traditional genetic heavy prescription jurisprudence in order to establish a stable and rapid genetic redistribution technique, and The identification of biologic phenotypic characteristics and vaccine safety and immunogenicity of the reA/SI/2006 (H1N1) Vca of the recombinant strain show that the donor strain A/Yunnan/1/2005Vca (H3N2) can be used as the mother parent of the live attenuated vaccine of influenza virus Vero cells. Two viruses are simultaneously infected with Vero cells, MDCK cells and chicken embryos, plus three cultures. The Sheep anti A/Yunnan/1/2005Vca (H3N2) virus sera neutralized donor strain and cultured in Vero cells at 25 degrees centigrade. Finally, the strains of reA/SI/2006 (H1N1) Vca., which can grow at 25 degrees centigrade in Vero cells, were successfully rematched in Vero cells and MDCK cells at 25 degrees centigrade, respectively. The results showed that the antigenicity of the redistribution strain was the same as that of the epidemic strain A/Solomom slands/3/2006 (H1N1). Further gene sequencing results showed that the reA/SI/2006 (H1N1) Vca surface antigen gene HA and NA were derived from the A/Solomom Islands/3/2006 (H1N1) of the epidemic strain, and the 6 internal genes were derived from the donor plant A/Yunnan/1/2005Vca. Under the culture conditions, reA/SI/2006 (H1N1) Vca was transmitted for 18 generations in Vero cells at 25 degrees centigrade, the virus titer was above 7.51gTCID50/mL and the virus type did not change during the passage. The infective titers of the reA/SI/2006 (H1N1) Vca at 25, 33 and 39 were 6.9,7.8 and 3.51gTCID50/mL respectively, with Ca, TS phenotype. 106 The viral load of ID50 was inoculated in BALB/c mice, and the viral load of the nasal clips and lung tissues was measured for 7 days. The viral load in the upper and lower respiratory tract was lower than 31gTCID50/g in the first five days of the reA/SI/2006 (H1N1) Vca group, indicating that the redistribution virus had a detoxification phenotype. 105TCID50106TCID50 and 107TCID50 three were used. BALB/c mice were immunized with two times, which could effectively stimulate the mice to produce the hemagglutination and neutralizing antibodies against the epidemic strain A/Solomom Islands/3/2006 (H1N1), and promote the secretion of sIgA. After inoculation of reA/SI/2006 (H1N1) Vca, the mice were not only protected from the same subtype influenza virus A/JN/2009 (H1N1). This study shows that reA/SI/2006 (H1N1) Vca is a kind of 50MLD50 (H1N1) Vca, which is obtained by genetic redistribution technology with both the surface antigen gene of the epidemic strain, and can also grow at 25 degrees centigrade in the Vero cell, and has the characteristic of the attenuated phenotype. Therefore, A/Yunnan/1/2005Vc A (H3N2) can be used as the maternal strain of influenza virus Vero cell cold adapted live attenuated vaccine.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 吳俊東;朱鳳才;;流感疫苗研究的進(jìn)展[J];江蘇預(yù)防醫(yī)學(xué);2008年02期

2 羅振武;邱豐;魏曉露;余磊;張?jiān)评?姚石寶;郭自全;唐成麗;姜述德;廖國(guó)陽(yáng);李衛(wèi)東;;流感病毒在Vero細(xì)胞上的適應(yīng)性[J];中國(guó)生物制品學(xué)雜志;2008年04期

3 劉錚;孫明波;高菁霞;馬磊;林小瑞;燕景恩;張勇;吳忠香;劉雅靈;姚忠萍;廖國(guó)陽(yáng);李衛(wèi)東;;低血清培養(yǎng)Vero細(xì)胞和流感病毒條件的優(yōu)化[J];中國(guó)生物制品學(xué)雜志;2009年06期

相關(guān)博士學(xué)位論文 前1條

1 楊景暉;H3N2亞型流感病毒Vero細(xì)胞冷適應(yīng)株減毒特性及假病毒評(píng)價(jià)中和抗體的研究[D];北京協(xié)和醫(yī)學(xué)院;2014年

，

本文編號(hào)：2115400