基于流感病毒HA2蛋白通用型疫苗的初步研究

发布时间：2018-03-03 08:12

本文选题：流感　切入点：通用型疫苗　出处：《中国疾病预防控制中心》2012年硕士论文　论文类型：学位论文

【摘要】：无论是流感大流行还是每年的季节性流感流行,都给人类健康带来巨大威胁,2009年甲型H1N1流感大流行再一次证明了这一点。流感疫苗接种是防控流感最重要的手段,由于流感病毒高度的变异性,流感疫苗的生产需要根据全球流感监测结果经常更换疫苗株,但由于目前对流感病毒变异预测的手段往往有限,往往会导致疫苗株与流行株不匹配,从而降低疫苗的保护率,通用型疫苗即能够对抗多种亚型流感病毒或流感变异株的疫苗,其保护效果不受流感病毒变异的影响,也不需要更换疫苗株,是目前流感疫苗研究领域的热点。本课题主要利用乙肝病毒核心抗原(Hepatitis B Core Antigen, HBc)蛋白可以形成病毒样颗粒(Virus Like Particle,VLP)的特点,将流感病毒的HA2的76-130AA线性保守区(Linear Conserved Region, LCR)与HBc融合表达,对所得到的VLP疫苗进行免疫学评价,希望能够得到一种可以提供交叉保护的流感通用型疫苗。本课题的研究目的： 1.利用HBc VLP载体平台表达流感病毒LCR,构建形成VLP疫苗。 2.对所构建的LCR-HBc VLP疫苗进行免疫学评价,通过动物实验研究其交叉保护效果。研究内容： 1.从A/Hubei/1/2010(H5N1)HA2基因序列出发,连同HBc基因按照大肠杆菌偏好的密码子进行优化,优化后的LCR-HBc基因全基因合成,将合成的基因插入到pET30a原核表达载体中,通过大肠杆菌原核系统表达该融合蛋白。 2.将经过Ni柱亲和层析纯化后的LCR-HBc VLP免疫小鼠,通过两次免疫后评价其HA特异性的抗体水平和IFN-y细胞免疫水平。利用H1N1鼠肺适应株PR8进行攻毒实验,检测其体重变化、肺病毒载量、肺病理切片和生存率等指标以评价其免疫效果和交叉保护作用。主要结果： 1.通过将优化后的LCR-HBc融合基因密码子全基因合成,LCR-HBc融合基因能在pET30a大肠杆菌原核表达系统中大量表达,并且经过纯化浓缩后,通过电镜观察证明可以正确组装形成VLP。 2.对以包涵体形式存在的LCR-HBc蛋白进行变形溶解后,利用其N端含有的6×His标签进行Ni柱亲和层析法纯化。结果表明其纯度可以达到85%以上。 3.小鼠经过两次免疫后,相比对照组,LCR-HBc疫苗组H5N1HA特异性IgG抗体具有显著升高,抗体滴度达到1：12800以上,表明LCR-HBc融合蛋白能够刺激机体产生高水平的HA特异性抗体。通过ELISPOT实验确定出了两条能够刺激小鼠脾淋巴细胞产生IFN-y的有效表位为：分别为位于HA280-89AA位置的LNKKMEDGFL以及120-129AA位置的DKVRLQLRDN,且经比对得知这两段序列在各个亚型中相对比较保守,提示LCR-HBc可能会刺激机体产生可针对许多亚型病毒的细胞免疫反应。 4.对免疫后小鼠的PR8致死剂量攻毒实验结果显示,LCR-HBc疫苗组体重改变较对照组低,肺病毒载量显著低于对照组(P0.01),肺病理切片结果表明疫苗组小鼠较对照组轻。疫苗组的保护率为50%。通过上述研究表明,流感病毒HA基因的LCR片段可以与HBC在大肠杆菌表达系统高效表达,并且可以组装成VLP颗粒。通过动物实验表明,LCR-HBc VLP疫苗可以产生很好的体液免疫和细胞免疫,对不同亚型流感病毒具有较好的交叉保护效果,为今后通用性疫苗的研究提供了一些启示。
[Abstract]:Whether it is an influenza pandemic or seasonal flu each year, all poses a great threat to human health, the 2009 H1N1 pandemic once again proved this point. Influenza vaccination is the most important means of prevention and control of influenza, because influenza virus highly variability, influenza vaccine production needs according to the monitoring results of influenza global frequent replacement but due to the current vaccine strains of influenza virus mutation prediction tools are often limited, often lead to the vaccine and the epidemic strains do not match, so as to reduce the vaccine protection rate, universal vaccine to influenza virus or influenza strains resistant to multiple vaccine, its protective effect against influenza virus variation. Do not need to replace the vaccine strain, influenza vaccine is currently a hot research field. This paper mainly use the hepatitis B virus core antigen (Hepatitis B Core Antigen, HBc). White can form virus like particles (Virus Like, Particle, VLP) the characteristics of influenza viruses HA2 76-130AA conserved region (Linear Conserved linear Region, LCR) and HBc fusion expression, immunological evaluation of the VLP vaccine, hoping to get a can provide cross protection of universal influenza vaccine.
The purpose of this study is as follows:
1. the HBc VLP carrier platform was used to express the influenza virus LCR, and the VLP vaccine was constructed.
2. the immunological evaluation of the constructed LCR-HBc VLP vaccine was carried out, and the effect of cross protection was studied by animal experiments.
Research content:
From 1. A/Hubei/1/2010 (H5N1) HA2 gene sequence of HBc gene in Escherichia coli, together with the preference of codon optimized gene LCR-HBc gene optimized synthesis, the synthetic gene was inserted into prokaryotic expression vector pET30a, the expression of the fusion protein by E.coli prokaryotic system.
2. through Ni affinity chromatography purified LCR-HBc VLP immunized mice, the specificity of HA was evaluated by two times after immunization the antibody level and IFN-y cell immunity. Using H1N1 mouse lung adapted strains of PR8 virus attack test, to detect the changes of body weight, lung viral load, lung pathology and survival rate to evaluate the immunogenicity and cross protection.
Main results:
1., by synthesizing the optimized LCR-HBc fusion gene codon full gene, LCR-HBc fusion gene can be expressed in pET30a Escherichia coli prokaryotic expression system. After purification and concentration, it can be correctly assembled to form VLP. by electron microscopy.
2., after the deformation and dissolution of the LCR-HBc protein existed in the inclusion body form, the Ni column affinity chromatography method was used to purify the N protein containing 6 x His tag. The result showed that its purity could reach 85%.
3. mice after two times of immunization, compared with control group, LCR-HBc vaccine group H5N1HA specific IgG antibody has significantly higher antibody titer reached more than 1:12800, showed that LCR-HBc fusion protein can stimulate the body to produce specific antibodies against HA high level by ELISPOT were determined in two effective form can stimulate the spleen lymphocytes of mice IFN-y a: were located in the HA280-89AA position of the LNKKMEDGFL and 120-129AA location of DKVRLQLRDN, and by comparing the two sequences in each subtype was relatively conservative, suggesting that LCR-HBc may stimulate the body to produce a cellular immune response in many subtypes of the virus.
4., the PR8 lethal dose test results of immunization mice showed that the weight change of LCR-HBc vaccine group was lower than that of the control group, and the load of lung virus was significantly lower than that of the control group (P0.01). The results of lung pathological section showed that the vaccine group was lighter than the control group. The protective rate of the vaccine group was 50%..
The results above indicated that the gene of influenza virus HA LCR fragment expression system and expression of HBC in Escherichia coli, and can be assembled into VLP particles. The animal experiment showed that LCR-HBc VLP vaccine can produce good humoral and cellular immunity, has good effect on the cross protection of different subtypes of influenza virus, provided some enlightenment for the future research on universal vaccines.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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