【摘要】： 【研究背景】 流感病毒(Influenza virus )是一種能夠引起嚴(yán)重性呼吸道傳播疾病的正粘科屬病毒。根據(jù)其內(nèi)部核蛋白(NP)和基質(zhì)蛋白(M)的抗原性不同,可分為甲(A)、乙(B)、丙(C)三種類型。其中,甲型流感病毒曾引起多次大范圍的流行,上世紀(jì)共出現(xiàn)四次大流行,最為嚴(yán)重的是1918年的大流感,造成了全世界5千多萬人死亡。2009年世界范圍內(nèi)暴發(fā)的豬源性甲型流感病毒流行,傳染性強(qiáng),波及范圍廣,再次突顯了甲型流感病毒感染的嚴(yán)重性及其對人類社會造成的巨大經(jīng)濟(jì)負(fù)擔(dān)。 接種疫苗是預(yù)防流感發(fā)生與傳播的最有效方式。目前市場應(yīng)用的流感疫苗主要有三種:全病毒滅活疫苗、裂解疫苗、亞單位疫苗。這些疫苗對同亞型流感病毒感染預(yù)防有效,但在不同亞型病毒之間的保護(hù)效果較弱。此外,流感病毒的變異速度非常快,流感疫苗每年都要更換,給疫苗的生產(chǎn)制備造成了諸多不便。世界衛(wèi)生組織是依據(jù)當(dāng)年全世界范圍流感病毒變化情況來預(yù)測并推薦下一年流感疫苗生產(chǎn)用組分的,預(yù)測的準(zhǔn)確度將會直接影響疫苗的保護(hù)效率,如預(yù)測失敗將造成流感爆發(fā)流行的潛在威脅。因此,研制出一種具有廣泛保護(hù)作用的通用型疫苗對于應(yīng)對流感病毒突變后的突然爆發(fā),迅速建立人群免疫屏障,阻斷流感大流行蔓延,降低其危害性方面具有極其重要的實際意義。 甲型流感通用疫苗的研發(fā)主要基于流感病毒基質(zhì)蛋白胞外功能區(qū)M2e (Ectodomain of matrix protein 2),其在各種甲型流感病毒中具有極高的保守性,且可誘導(dǎo)特異性保護(hù)抗體產(chǎn)生。此外還有高度保守的流感病毒核蛋白NP(Nucleoprotein),其表面具有多個細(xì)胞毒性T淋巴細(xì)胞(CTL)識別表位,可誘導(dǎo)細(xì)胞免疫。但M2e和NP表位的分子量小、免疫原性低、易降解,在體內(nèi)難以產(chǎn)生足夠的免疫反應(yīng)。所以,使用結(jié)構(gòu)復(fù)雜病毒蛋白作為免疫原性放大載體成為表位疫苗研發(fā)的熱點。其中,乙肝病毒核心蛋白HBc(Hepatitis B virus core protein , HBc)蛋白具有天然的顆粒組裝能力,能特異性激發(fā)針對外源表位的體液、細(xì)胞免疫。近年來,HBc顆粒的應(yīng)用方面已經(jīng)有了較成熟研究結(jié)果。疫苗研究中以HBc作為載體的疫苗抗原有很多,如乙肝病毒表面抗原、人乳頭瘤病毒E7蛋白、B群鏈球菌CPS糖蛋白等,在臨床前實驗研究中已觀察到肯定的療效。因此,發(fā)展以HBc顆粒為載體的流感病毒表位疫苗研究具有很好的前景和應(yīng)用價值。 【目的】 本研究的目的是構(gòu)建以乙肝病毒核心蛋白(HBc)為載體的基于M2e蛋白的甲型流感通用疫苗抗原,利用桿狀病毒表達(dá)系統(tǒng)及大腸桿菌表達(dá)系統(tǒng)表達(dá)、純化和鑒定,并進(jìn)行疫苗免疫效果初步評價,為發(fā)展安全、有效、具有廣泛保護(hù)作用的甲型流感通用疫苗奠定基礎(chǔ)。 【方法與結(jié)果】 第一部分sf9昆蟲細(xì)胞表達(dá)甲型流感M2e通用疫苗抗原的構(gòu)建、表達(dá)、純化及鑒定通過基因工程方法,構(gòu)建含M2e重復(fù)片段的3M2e-HBc-pFastBacHTA重組質(zhì)粒。將該重組質(zhì)粒與DH10Bac中的穿梭載體Bacmid同源重組,獲得3M2e-HBc-Bacmid重組桿粒。提取桿粒DNA轉(zhuǎn)染sf9昆蟲細(xì)胞,得到含有重組桿狀病毒。繼續(xù)擴(kuò)增病毒,感染細(xì)胞后,免疫熒光檢測成功表達(dá)出3M2e-HBc目的蛋白。Western blot檢測該蛋白具有免疫活性。利用pFastBacHTA載體上帶有的組氨酸標(biāo)簽,進(jìn)行親和層析,純化獲得3M2e-HBc重組蛋白。電鏡結(jié)果顯示,3M2e-HBc目的基因可以通過桿狀病毒sf9昆蟲細(xì)胞表達(dá)系統(tǒng)表達(dá)并正確裝配出病毒樣顆粒。 第二部分大腸桿菌表達(dá)甲型流感M2e-NP通用疫苗抗原的構(gòu)建、表達(dá)、純化及鑒定通過基因工程方法,構(gòu)建并鑒定獲得3M2e-NP-HBc-pET 21a重組表達(dá)質(zhì)粒,并轉(zhuǎn)化大腸桿菌細(xì)胞,IPTG誘導(dǎo)表達(dá)首先獲得了包涵體形式表達(dá)的重組蛋白。經(jīng)變性及復(fù)性純化處理后,包涵體蛋白形成的重組病毒顆粒的狀態(tài)不理想。經(jīng)進(jìn)一步優(yōu)化誘導(dǎo)表達(dá)條件獲得了部分可溶性表達(dá)的重組蛋白,Western blot檢測證明該蛋白具有免疫活性。采用層析方法獲得純化的3M2e-NP-HBc重組蛋白。電鏡觀察結(jié)果顯示,大腸桿菌表達(dá)的可溶性重組蛋白能夠自動裝配成大小約為30nm的病毒樣顆粒。 第三部分甲型流感通用疫苗免疫效果初步評價 通過動物免疫實驗,初步評價甲型流感通用疫苗的免疫應(yīng)答效果及交叉保護(hù)作用。采用間接ELISA法、流式細(xì)胞術(shù)、ELISOPT技術(shù),對疫苗在Balb/C小鼠體內(nèi)的免疫應(yīng)答效果進(jìn)行了評估。在對大腸桿菌表達(dá)的流感通用疫苗評價的結(jié)果中顯示,疫苗以滴鼻和腹腔注射兩種途徑免疫小鼠,均可以誘導(dǎo)產(chǎn)生針對不同亞型流感病毒的特異性體液及細(xì)胞免疫應(yīng)答,并且滴鼻免疫能夠激發(fā)更高的黏膜局部免疫應(yīng)答,說明采用滴鼻免疫這種更接近于流感病毒自然感染途徑的方式免疫對提高流感疫苗的保護(hù)效果具有重要意義。同時,攻毒試驗結(jié)果顯示,以A/Beijing/501和A/PR/8/34兩株代表性的甲型流感病毒攻毒后,疫苗免疫組相比對照組明顯降低小鼠的死亡率,表明大腸桿菌表達(dá)的以HBc為載體的甲型流感通用疫苗具有良好的免疫原性交叉保護(hù)作用。 【結(jié)論】 本研究利用Bac-to-Bac桿狀病毒sf9昆蟲細(xì)胞表達(dá)系統(tǒng)和大腸桿菌原核表達(dá)系統(tǒng),分別成功制備了兩種的以HBc為載體的基于流感病毒M2e蛋白的流感通用疫苗,并主要對大腸桿菌表達(dá)的流感通用疫苗進(jìn)行了動物免疫效果評價。實驗結(jié)果顯示,該疫苗具有很好的免疫原性,可以刺激機(jī)體產(chǎn)生系統(tǒng)免疫應(yīng)答和黏膜局部免疫應(yīng)答,并且能產(chǎn)生有效的交叉保護(hù)作用,為甲型流感通用疫苗的深入研究提供了理論依據(jù)。
[Abstract]:[background]
Influenza virus (Influenza virus) is a kind of Orthomyxovirus which can cause severe respiratory diseases. According to the antigenicity of nucleoprotein (NP) and matrix protein (M), Influenza virus can be divided into three types: A (A), B (B) and C (C). Influenza A virus has caused a wide range of pandemics, and there were four major outbreaks in the last century. What is most serious is the large influenza in 1918, which resulted in the death of more than 5 million people worldwide. The outbreak of swine influenza A * virus in the world in.2009 was epidemic, infectious and spread widely. It once again highlighted the seriousness of influenza A virus infection and its enormous economic burden on human society.
Vaccination is the most effective way to prevent the occurrence and spread of influenza. At present, there are three kinds of influenza vaccines in the market: whole virus inactivated vaccine, lysis vaccine and subunit vaccine. The World Health Organization (WHO) predicts and recommends the components for the next year's influenza vaccine production based on changes in influenza viruses worldwide. The accuracy of the prediction will directly affect the efficiency of vaccine protection, such as the failure of the prediction. Therefore, it is of great practical significance to develop a universal vaccine with extensive protective effect in dealing with the sudden outbreak of influenza virus mutation, rapidly establishing a population immune barrier, blocking the spread of influenza pandemic and reducing its harmfulness.
The development of universal influenza A vaccine is mainly based on the extracellular domain M2e (Ectodomain of matrix protein 2) of influenza virus, which is highly conserved among influenza viruses and can induce the production of specific protective antibodies. Multiple cytotoxic T lymphocyte (CTL) epitopes can induce cellular immunity. However, M2e and NP epitopes are small in molecular weight, low in immunogenicity, easy to degrade, and difficult to produce sufficient immune response in vivo. Therefore, the use of complex viral proteins as immunogenic amplification vectors has become a hot topic in epitope vaccine research and development. Hepatitis B virus core protein (HBc) has a natural ability to assemble particles, which can specifically stimulate humoral and cellular immunity against exogenous epitopes. In recent years, there have been more mature research results in the application of HBc particles. Pro-HPV E7 protein, group B streptococcal CPS glycoprotein and so on have been observed to be effective in preclinical studies. Therefore, the development of influenza virus epitope vaccine with HBc particles as carriers has a good prospect and application value.
[Objective]
The aim of this study is to construct a universal influenza A vaccine antigen based on M2e protein with hepatitis B virus core protein (HBc) as carrier, express, purify and identify the influenza A vaccine antigen by baculovirus expression system and E. coli expression system, and preliminarily evaluate the immune effect of the vaccine, so as to develop a safe, effective and widely protective influenza A vaccine. The universal vaccine is the foundation.
[methods and results]
The first part is the construction, expression, purification and characterization of the M2e vaccine antigen expressed by Sf9 insect cells. The 3M2e-HBc-pFastBacHTA recombinant plasmid containing M2e repeat fragment was constructed by genetic engineering. The recombinant plasmid was homologously recombined with the shuttle vector Bacmid in DH10Bac to obtain 3M2e-HBc-Bacmid recombinant plasmid. The recombinant protein 3M2e-HBc was successfully expressed by immunofluorescence assay. Western blot assay showed that the protein was immunocompetent. The recombinant protein 3M2e-HBc was purified by affinity chromatography with the histidine tag on the pFastBacHTA vector. The results showed that the target gene of 3M2e-HBc could be expressed in baculovirus Sf9 insect cell expression system and assembled into virus-like particles correctly.
In the second part, the recombinant expression plasmid 3M2e-NP-HBc-pET 21a was constructed and identified by genetic engineering and transformed into E.coli cells. The recombinant protein expressed in the form of inclusion body was first obtained by IPTG induction. The recombinant protein of 3M2e-NP-HBc was purified by chromatography. Electron microscopy showed that the recombinant protein of 3M2e-NP-HBc was purified from the colon stem. The soluble recombinant protein expressed by the bacteria can be automatically assembled into a virus like particle size of about 30nm.
The third part is a preliminary evaluation of the immune effect of universal influenza A vaccine.
The immune response and cross-protection of the universal influenza A vaccine in Balb/C mice were preliminarily evaluated by animal immune test. The immune response of the vaccine in Balb/C mice was evaluated by indirect ELISA, flow cytometry and ELISOPT techniques. Both intranasal and intraperitoneal immunization can induce specific humoral and cellular immune responses to different subtypes of influenza viruses, and intranasal immunization can stimulate higher local mucosal immune responses, suggesting that intranasal immunization is a more natural route for influenza virus infection. The protective effect of high influenza vaccine is very important. Meanwhile, the results of virus attack test showed that the mortality of mice in the vaccine immunized group was significantly lower than that in the control group after two typical strains of influenza A virus (A/Beijing/501 and A/PR/8/34) were attacked. Immunogenic cross protection.
[Conclusion]
In this study, Bac-to-Bac baculovirus Sf9 insect cell expression system and E. The vaccine has good immunogenicity, can stimulate the body to produce systemic immune response and local mucosal immune response, and can produce effective cross-protection, which provides a theoretical basis for the further study of influenza A vaccine.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.1

【共引文獻(xiàn)】

相關(guān)期刊論文 前2條

1 戚鳳春,盛軍;流感疫苗的研究進(jìn)展[J];中國生物制品學(xué)雜志;2004年03期

2 王新衛(wèi);畢英佐;何宏軒;王憲文;詹愛軍;馬靜云;;AIV NP基因在噬菌體表面的展示及間接ELISA方法的建立[J];畜牧獸醫(yī)學(xué)報;2008年11期

相關(guān)博士學(xué)位論文 前1條

1 胡思順;禽流感病毒H9N2亞型和雞毒霉形體HS株主要抗原性基因的克隆與表達(dá)研究[D];華中農(nóng)業(yè)大學(xué);2005年

相關(guān)碩士學(xué)位論文 前6條

1 孫愛軍;禽流感二價油乳劑滅活苗的研究[D];吉林農(nóng)業(yè)大學(xué);2003年

2 杜麗娟;流感裂解滅活疫苗純化工藝的開發(fā)[D];大連理工大學(xué);2006年

3 孫穎;A型流感病毒HA DNA疫苗和原核表達(dá)載體的構(gòu)建、表達(dá)及多克隆抗體制備[D];東北師范大學(xué);2008年

4 吳淑春;禽流感四通道絲網(wǎng)印刷碳電極酶免疫傳感器的研制[D];浙江工商大學(xué);2008年

5 劉蕊;以HBC和HPV16L1為載體的流感通用疫苗研究[D];第四軍醫(yī)大學(xué);2008年

6 何雯雯;禽流感病毒HA2合成肽與M2e重組蛋白免疫保護(hù)作用的初步研究[D];安徽農(nóng)業(yè)大學(xué);2013年

，

本文編號：2229479