本文選題：流感病毒 + 反向遺傳學技術(shù)　； 參考：《西北農(nóng)林科技大學》2008年碩士論文

【摘要】： 流行性感冒(Influence)是由流感病毒(influenza virus)引起的一種急性、接觸性呼吸道疾病。流感病毒屬于正黏病毒科(Orthomyxoviridae),負鏈RNA分節(jié)段病毒。根據(jù)保守的內(nèi)部蛋白(主要為NP和基質(zhì)蛋白M)的血清學反應(yīng)將流感病毒分為甲(A)、乙(B)、丙(C)三型。流感病毒可以感染包括人、馬、豬、水生哺乳動物和禽類在內(nèi)的多種動物。二十世紀四次大流行和近年來突破種間屏障高致病性禽流感的發(fā)生嚴重影響了人類社會安定,并引起巨大的經(jīng)濟損失。近年來,隨著我國人流感流行規(guī)律的變化,發(fā)病率上升,使流感的防制越來越重要。 當前流感疫苗是防治流感的主要手段。五十多年來,流感疫苗制備的方法得到了很大的發(fā)展。尤其近年來,以冷適應(yīng)減毒株為背景,反向遺傳學技術(shù)為基礎(chǔ),拯救重配流感病毒弱毒活疫苗株,進而研發(fā)新一代的流感弱毒活疫苗成為當今流感疫苗研究的熱點。 本研究基于本實驗室構(gòu)建的冷適應(yīng)病毒拯救系統(tǒng),利用反向遺傳學技術(shù),將冷適應(yīng)流感病毒株A/Ann Arbor/6/60 (H2N2)的六個內(nèi)部基因和WHO公布的2006～2007年A型流感病毒流行株A/New Caledonia/20/99(H1N1)的兩個外部基因進行了重配,拯救出既具有冷適應(yīng)和溫度敏感表型,又具有流行株抗原性的重配病毒rMDV-H1。以重配病毒為抗原,通過鼻腔途徑免疫小鼠,測定了小鼠血清的血凝抑制抗體,血清IgG,進行IgG亞類分析和鼻、肺及陰道沖洗液中sIgA,從而對重配病毒的免疫原性進行了初步的鑒定,為我國流感弱毒疫苗的研究提供一定的依據(jù)。 1. 2006—2007流行株A/New Caledonia/20/99(H1N1)冷適應(yīng)株rMDV-H1的拯救。RT-PCR擴增A/New Caledonia/20/99(H1N1)的HA和NA基因,連接pMD-19T載體并測序,將序列正確的HA和NA基因酶切后克隆至雙向轉(zhuǎn)錄/表達載體pAD3000上,分別與帶有PR8的六個內(nèi)部基因的質(zhì)粒共轉(zhuǎn)染COS-1細胞,33℃、5% CO2培養(yǎng)48 h,轉(zhuǎn)染上清液接種10日齡SPF雞胚,96 h后檢測雞胚尿囊液血凝效價,得到有血凝效價的“7+1”重配病毒,驗證了pMVD-H1HA、pMVD-H1NA的功能。 將驗證好的質(zhì)粒pMVD-H1HA、pMVD-H1NA分別與帶有冷適應(yīng)株的六個內(nèi)部基因的質(zhì)粒共轉(zhuǎn)染COS-1細胞,33℃、5%CO2培養(yǎng)48 h,轉(zhuǎn)染上清液接種10日齡SPF雞胚,96 h后檢測雞胚尿囊液血凝效價,得到有血凝性的重組病毒,即rMDV-H1。 2.冷適應(yīng)候選株rMDV-H1的生物學特性的初步鑒定。 對重組病毒rMDV-H1的生物學特性進行初步研究。包括穩(wěn)定性試驗,細胞病變(CPE)的觀察,間接免疫熒光試驗(IFA),使用雞胚半數(shù)感染量(EID50)和半數(shù)組織感染量(TCID50)進行毒力的測定。 3.冷適應(yīng)候選株rMDV-H1免疫原性的初步研究。 將rMDV-H1第四代雞胚尿囊液接種10日齡SPF雞胚100枚(0.25mL/胚),33℃孵育72 h,收獲血凝效價≥28的雞胚尿囊液,經(jīng)超高速蔗糖梯度離心法濃縮純化抗原。使用雞胚半數(shù)感染量(EID50)和半數(shù)組織感染量(TCID50)測定純化后抗原的毒力。根據(jù)EID50和TCID50的實驗結(jié)果,將20只6～8周齡的Balb/c雌性小鼠分為四組,每組5只,接種抗原量依次為0、0.05EID50、0.1EID50和0.05EID50。接種途徑分為皮下注射和鼻腔接種。第1次免疫后14 d加強免疫1次。采集第2次免疫后7 d、14 d血清,經(jīng)HIA試驗和間接免疫酶聯(lián)吸附試驗(ELISA)分別測定血凝抑制抗體和特異性血清抗體IgG,并進行IgG1、IgG2a和IgG2b亞類分析。采集第2次免疫后14 d肺、鼻和陰道沖洗液,經(jīng)ELISA測定局部分泌性sIgA。
[Abstract]:Influenza A (Influence) influenza virus (influenza virus) caused an acute, contagious respiratory disease. The influenza virus belongs to the Orthomyxoviridae (Orthomyxoviridae), negative stranded RNA virus. According to the segmental conserved internal proteins (mainly NP and matrix protein M) serological response to influenza viruses divided into a (A), B (B), C (C) type three. Influenza virus can infect human beings, horses, pigs, poultry and aquatic mammals including a variety of animal. In twentieth Century four pandemics occurred in recent years and break the species barrier of highly pathogenic avian influenza has seriously affected the social stability. And caused huge economic losses. In recent years, with the change of China's human influenza epidemic regularity, the rising incidence of influenza prevention, make more and more important.
The current flu vaccine is the main means of prevention and treatment of influenza. Over the past fifty years, the influenza vaccine preparation method has been greatly developed. Especially in recent years, the cold adapted attenuated as the background, the reverse genetics technology as the foundation, save the reassortant influenza virus live attenuated vaccine strain, and to develop a new generation of attenuated influenza the vaccine has become the hotspot of influenza vaccine research.
This study constructed in our laboratory based on cold adapted virus rescue system, using reverse genetics technology, the cold adapted influenza virus strain A/Ann Arbor/6/60 (H2N2) six internal genes and WHO released 2006~2007 years influenza A virus strain A/New Caledonia/20/99 (H1N1) of the two external gene was carried out with heavy, save as with cold adaptation and temperature sensitive phenotype, with reassortant virus rMDV-H1. strains with the antigenicity of reassortant virus as antigen immunized mice by the nasal route, hemagglutination inhibition antibody in mice serum were measured, serum IgG, IgG subgroup analysis and nasal, lung and vaginal lavage fluid in sIgA, so as to re distribution the immunogenicity of the virus was preliminarily identified as China's weak research provide a basis for influenza virus vaccine.
1.2006 - 2007 strains A/New Caledonia/20/99 (H1N1) cold adapted strain rMDV-H1 rescue.RT-PCR Caledonia/20/99 amplification of A/New (H1N1) HA and NA gene, connected to pMD-19T vector and sequenced, the correct sequence of HA and NA gene digested and cloned into bidirectional transcription / expression vector pAD3000, respectively, and six internal genes with plasmid the PR8 were co transfected into COS-1 cells, 33 C, 5% CO2 48 h culture, the transfected supernatants were inoculated into 10 day old SPF chicken embryo allantoic fluid detection, hemagglutination titer of 96 h, with the hemagglutination titer of the "7+1" reassortant virus, verify the pMVD-H1HA pMVD-H1NA function.
Plasmid pMVD-H1HA will verify the pMVD-H1NA and plasmids with cold adapted strains of six internal genes were transfected into COS-1 cells, 33 C, 5%CO2 and cultured for 48 h, the transfected supernatants were inoculated into 10 day old SPF chicken embryo allantoic fluid detection, hemagglutination titer of 96 h, with recombinant virus hemagglutination, i.e. rMDV-H1.
2. preliminary identification of the biological characteristics of the 2. cold adaptable candidate strain.
The biological characteristics of recombinant virus rMDV-H1 were preliminarily studied, including stability test, cytopathic (CPE) observation, indirect immunofluorescence test (IFA), and the detection of virulence by using chicken embryo half infection (EID50) and half tissue infection (TCID50).
A preliminary study on the immunogenicity of 3. cold adapted candidate strain rMDV-H1.
The fourth generation of rMDV-H1 allantoic fluid of 10 day old SPF chicken 100 (0.25mL/ embryo) were incubated for 72 h, 33 DEG C, the hemagglutination titer of allantoic fluid harvested more than 28, with super high speed sucrose gradient centrifugation. The purified antigen concentration using eid50 (EID50) and tissue infection dose (TCID50 determination of toxicity of the purified antigen). According to the experimental results of EID50 and TCID50, 20 6~8 week old female Balb/c mice were divided into four groups, 5 rats in each group, inoculation amount of antigen were 0,0.05EID50,0.1EID50 and 0.05EID50. inoculation way divided into subcutaneous and intranasal inoculation. After the first immunization, 14 d immunization for 1 times. Collected after the second immunization, 7 d, 14 d of serum, by HIA test and indirect enzyme linked immunosorbent assay (ELISA) were determined by hemagglutination inhibition antibody and specific serum antibody and IgG, IgG1, IgG2a and IgG2b subgroup analysis. Collected after the second immunization, 14 d of lung, nasal and Yin DDF lotion and partial secreting sIgA. by ELISA

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R373.1

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