本文選題：流感　切入點：通用型疫苗　出處：《中國疾病預(yù)防控制中心》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：無論是流感大流行還是每年的季節(jié)性流感流行,都給人類健康帶來巨大威脅,2009年甲型H1N1流感大流行再一次證明了這一點。流感疫苗接種是防控流感最重要的手段,由于流感病毒高度的變異性,流感疫苗的生產(chǎn)需要根據(jù)全球流感監(jiān)測結(jié)果經(jīng)常更換疫苗株,但由于目前對流感病毒變異預(yù)測的手段往往有限,往往會導(dǎo)致疫苗株與流行株不匹配,從而降低疫苗的保護率,通用型疫苗即能夠?qū)苟喾N亞型流感病毒或流感變異株的疫苗,其保護效果不受流感病毒變異的影響,也不需要更換疫苗株,是目前流感疫苗研究領(lǐng)域的熱點。本課題主要利用乙肝病毒核心抗原(Hepatitis B Core Antigen, HBc)蛋白可以形成病毒樣顆粒(Virus Like Particle,VLP)的特點,將流感病毒的HA2的76-130AA線性保守區(qū)(Linear Conserved Region, LCR)與HBc融合表達(dá),對所得到的VLP疫苗進(jìn)行免疫學(xué)評價,希望能夠得到一種可以提供交叉保護的流感通用型疫苗。 本課題的研究目的： 1.利用HBc VLP載體平臺表達(dá)流感病毒LCR,構(gòu)建形成VLP疫苗。 2.對所構(gòu)建的LCR-HBc VLP疫苗進(jìn)行免疫學(xué)評價,通過動物實驗研究其交叉保護效果。 研究內(nèi)容： 1.從A/Hubei/1/2010(H5N1)HA2基因序列出發(fā),連同HBc基因按照大腸桿菌偏好的密碼子進(jìn)行優(yōu)化,優(yōu)化后的LCR-HBc基因全基因合成,將合成的基因插入到pET30a原核表達(dá)載體中,通過大腸桿菌原核系統(tǒng)表達(dá)該融合蛋白。 2.將經(jīng)過Ni柱親和層析純化后的LCR-HBc VLP免疫小鼠,通過兩次免疫后評價其HA特異性的抗體水平和IFN-y細(xì)胞免疫水平。利用H1N1鼠肺適應(yīng)株P(guān)R8進(jìn)行攻毒實驗,檢測其體重變化、肺病毒載量、肺病理切片和生存率等指標(biāo)以評價其免疫效果和交叉保護作用。 主要結(jié)果： 1.通過將優(yōu)化后的LCR-HBc融合基因密碼子全基因合成,LCR-HBc融合基因能在pET30a大腸桿菌原核表達(dá)系統(tǒng)中大量表達(dá),并且經(jīng)過純化濃縮后,通過電鏡觀察證明可以正確組裝形成VLP。 2.對以包涵體形式存在的LCR-HBc蛋白進(jìn)行變形溶解后,利用其N端含有的6×His標(biāo)簽進(jìn)行Ni柱親和層析法純化。結(jié)果表明其純度可以達(dá)到85%以上。 3.小鼠經(jīng)過兩次免疫后,相比對照組,LCR-HBc疫苗組H5N1HA特異性IgG抗體具有顯著升高,抗體滴度達(dá)到1：12800以上,表明LCR-HBc融合蛋白能夠刺激機體產(chǎn)生高水平的HA特異性抗體。通過ELISPOT實驗確定出了兩條能夠刺激小鼠脾淋巴細(xì)胞產(chǎn)生IFN-y的有效表位為：分別為位于HA280-89AA位置的LNKKMEDGFL以及120-129AA位置的DKVRLQLRDN,且經(jīng)比對得知這兩段序列在各個亞型中相對比較保守,提示LCR-HBc可能會刺激機體產(chǎn)生可針對許多亞型病毒的細(xì)胞免疫反應(yīng)。 4.對免疫后小鼠的PR8致死劑量攻毒實驗結(jié)果顯示,LCR-HBc疫苗組體重改變較對照組低,肺病毒載量顯著低于對照組(P0.01),肺病理切片結(jié)果表明疫苗組小鼠較對照組輕。疫苗組的保護率為50%。 通過上述研究表明,流感病毒HA基因的LCR片段可以與HBC在大腸桿菌表達(dá)系統(tǒng)高效表達(dá),并且可以組裝成VLP顆粒。通過動物實驗表明,LCR-HBc VLP疫苗可以產(chǎn)生很好的體液免疫和細(xì)胞免疫,對不同亞型流感病毒具有較好的交叉保護效果,為今后通用性疫苗的研究提供了一些啟示。
[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.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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本文編號：1560219