A型流感病毒冷适应株的拯救及免疫原性的初步研究

发布时间：2018-04-15 00:35

本文选题：流感病毒 + 反向遗传学技术　；参考：《西北农林科技大学》2008年硕士论文

【摘要】： 流行性感冒(Influence)是由流感病毒(influenza virus)引起的一种急性、接触性呼吸道疾病。流感病毒属于正黏病毒科(Orthomyxoviridae),负链RNA分节段病毒。根据保守的内部蛋白(主要为NP和基质蛋白M)的血清学反应将流感病毒分为甲(A)、乙(B)、丙(C)三型。流感病毒可以感染包括人、马、猪、水生哺乳动物和禽类在内的多种动物。二十世纪四次大流行和近年来突破种间屏障高致病性禽流感的发生严重影响了人类社会安定,并引起巨大的经济损失。近年来,随着我国人流感流行规律的变化,发病率上升,使流感的防制越来越重要。当前流感疫苗是防治流感的主要手段。五十多年来,流感疫苗制备的方法得到了很大的发展。尤其近年来,以冷适应减毒株为背景,反向遗传学技术为基础,拯救重配流感病毒弱毒活疫苗株,进而研发新一代的流感弱毒活疫苗成为当今流感疫苗研究的热点。本研究基于本实验室构建的冷适应病毒拯救系统,利用反向遗传学技术,将冷适应流感病毒株A/Ann Arbor/6/60 (H2N2)的六个内部基因和WHO公布的2006～2007年A型流感病毒流行株A/New Caledonia/20/99(H1N1)的两个外部基因进行了重配,拯救出既具有冷适应和温度敏感表型,又具有流行株抗原性的重配病毒rMDV-H1。以重配病毒为抗原,通过鼻腔途径免疫小鼠,测定了小鼠血清的血凝抑制抗体,血清IgG,进行IgG亚类分析和鼻、肺及阴道冲洗液中sIgA,从而对重配病毒的免疫原性进行了初步的鉴定,为我国流感弱毒疫苗的研究提供一定的依据。 1. 2006—2007流行株A/New Caledonia/20/99(H1N1)冷适应株rMDV-H1的拯救。RT-PCR扩增A/New Caledonia/20/99(H1N1)的HA和NA基因,连接pMD-19T载体并测序,将序列正确的HA和NA基因酶切后克隆至双向转录/表达载体pAD3000上,分别与带有PR8的六个内部基因的质粒共转染COS-1细胞,33℃、5% CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96 h后检测鸡胚尿囊液血凝效价,得到有血凝效价的“7+1”重配病毒,验证了pMVD-H1HA、pMVD-H1NA的功能。将验证好的质粒pMVD-H1HA、pMVD-H1NA分别与带有冷适应株的六个内部基因的质粒共转染COS-1细胞,33℃、5%CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96 h后检测鸡胚尿囊液血凝效价,得到有血凝性的重组病毒,即rMDV-H1。 2.冷适应候选株rMDV-H1的生物学特性的初步鉴定。对重组病毒rMDV-H1的生物学特性进行初步研究。包括稳定性试验,细胞病变(CPE)的观察,间接免疫荧光试验(IFA),使用鸡胚半数感染量(EID50)和半数组织感染量(TCID50)进行毒力的测定。 3.冷适应候选株rMDV-H1免疫原性的初步研究。将rMDV-H1第四代鸡胚尿囊液接种10日龄SPF鸡胚100枚(0.25mL/胚),33℃孵育72 h,收获血凝效价≥28的鸡胚尿囊液,经超高速蔗糖梯度离心法浓缩纯化抗原。使用鸡胚半数感染量(EID50)和半数组织感染量(TCID50)测定纯化后抗原的毒力。根据EID50和TCID50的实验结果,将20只6～8周龄的Balb/c雌性小鼠分为四组,每组5只,接种抗原量依次为0、0.05EID50、0.1EID50和0.05EID50。接种途径分为皮下注射和鼻腔接种。第1次免疫后14 d加强免疫1次。采集第2次免疫后7 d、14 d血清,经HIA试验和间接免疫酶联吸附试验(ELISA)分别测定血凝抑制抗体和特异性血清抗体IgG,并进行IgG1、IgG2a和IgG2b亚类分析。采集第2次免疫后14 d肺、鼻和阴道冲洗液,经ELISA测定局部分泌性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

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373.1

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