新型甲型H1N1流感病毒血凝素蛋白人源化中和單抗和多肽表位疫苗研究
發(fā)布時(shí)間：2018-08-31 17:16
【摘要】：流感病毒作為最主要人類(lèi)感染病毒之一,對(duì)人類(lèi)的危害巨大。曾在上個(gè)世紀(jì)引起了三次全球性的流感大流行,造成數(shù)千萬(wàn)人的死亡。進(jìn)入新世紀(jì)以來(lái),在2009年爆發(fā)了全球性的流感大流行,給世界的社會(huì)與經(jīng)濟(jì)發(fā)展造成了一定程度的負(fù)面影響。 盡管已經(jīng)研發(fā)出滅活流感疫苗,較好的控制了該病毒株的持續(xù)流行和毒性加劇。但是,目前針對(duì)新型甲型H1N1流感病毒的免疫學(xué)及其致病機(jī)理仍了解不多。血凝素(haemegglutinin, HA),作為最主要的參與病毒感染的表面抗原之一,對(duì)于H1N1病毒的研究具有重要意義。 本研究利用桿狀病毒昆蟲(chóng)表達(dá)系統(tǒng)表達(dá)了具有生物學(xué)活性的新型甲型H1N1 HA蛋白和NA蛋白,以此為基礎(chǔ),開(kāi)展了如下兩方面研究： 首先,開(kāi)展了新型甲型H1N1 HA蛋白人中和單克隆抗體的制備。利用EBV轉(zhuǎn)化記憶性B細(xì)胞,在CpG和飼養(yǎng)細(xì)胞的存在下,培養(yǎng)轉(zhuǎn)化B細(xì)胞；經(jīng)有限稀釋后篩選能分泌HA蛋白特異性抗體的細(xì)胞克隆,共篩選96U孔板135塊包含12960株B細(xì)胞樣品；最終,經(jīng)多次篩選獲得45株陽(yáng)性克隆細(xì)胞。這些陽(yáng)性細(xì)胞克隆中有36株陽(yáng)性細(xì)胞克隆在隨后1-2月中死亡,提示EBV轉(zhuǎn)化的B細(xì)胞中絕大部分是短壽命的,而僅有少數(shù)才能長(zhǎng)期存活(2月)。為避免陽(yáng)性克隆細(xì)胞進(jìn)一步丟失,我們將陽(yáng)性細(xì)胞與K6H6/B5細(xì)胞系進(jìn)行PEG融合以獲得永生的陽(yáng)性細(xì)胞。然而經(jīng)多次實(shí)驗(yàn),但卻無(wú)法篩選到雜交瘤細(xì)胞。在不能獲得永生陽(yáng)性細(xì)胞的情況下,本研究改變研究策略,通過(guò)單細(xì)胞克隆團(tuán)5'-RACE方法獲得4對(duì)陽(yáng)性細(xì)胞輕、重鏈可變區(qū)cDNA基因,進(jìn)而利用桿狀病毒昆蟲(chóng)細(xì)胞表達(dá)該基因工程抗體。結(jié)果表明,昆蟲(chóng)細(xì)胞不是基因工程抗體表達(dá)的合適宿主細(xì)胞,盡管本研究在采用昆蟲(chóng)表達(dá)時(shí)也有文獻(xiàn)支持。下一步研究中需要使用更合適的CHO細(xì)胞表達(dá)上述HA特異性人基因工程抗體。 其次,本課題進(jìn)行了新型甲型流感病毒HA表位肽疫苗研究。流感病毒利用連續(xù)點(diǎn)突變和基因重組導(dǎo)致不斷變異,進(jìn)而逃避機(jī)體的記憶免疫應(yīng)答。目前的疫苗往往只是針對(duì)某一特定的病毒株或亞型,不能保護(hù)機(jī)體不被其他病毒株感染。而針對(duì)表位疫苗的設(shè)計(jì),通常利用的是線性表位,無(wú)論是B細(xì)胞表位,還是T細(xì)胞表位。本部分課題通過(guò)設(shè)計(jì)能模擬構(gòu)象表位的肽疫苗,進(jìn)而免疫小鼠,利用ELISA、血凝抑制實(shí)驗(yàn)、中和實(shí)驗(yàn)以及肽阻斷ELISA實(shí)驗(yàn),評(píng)價(jià)其是否能夠引起抗體免疫應(yīng)答。結(jié)果顯示,該HA肽疫苗免疫接種的小鼠血清能夠結(jié)合肽、重組H1N1 HA蛋白、重組H5N1 HA蛋白以及H1N1滅活疫苗；能夠抑制血凝實(shí)驗(yàn),同時(shí)還能夠中和非同種流感病毒亞型的H1N1和H5N1病毒。上述結(jié)果表明,新型HA肽疫苗能夠誘導(dǎo)機(jī)體產(chǎn)生相應(yīng)的體液免疫應(yīng)答,并且具有交叉保護(hù)作用。 綜上所述,本研究取得了以下主要成果： 1、利用桿狀病毒表達(dá)系統(tǒng)成功表達(dá)出具有生物學(xué)活性的HA蛋白和NA蛋白,對(duì)BaculoGold system和Bac-to-Bac system這兩種桿狀病毒昆蟲(chóng)表達(dá)系統(tǒng)進(jìn)行比較,結(jié)果表明后者更適合表達(dá)HA蛋白。 2、EBV轉(zhuǎn)化B細(xì)胞,篩選到9株能夠分泌HA特異性抗體的B細(xì)胞,進(jìn)一步獲得4對(duì)陽(yáng)性細(xì)胞輕、重鏈可變區(qū)cDNA基因,為進(jìn)一步制備基因工程抗體打下了基礎(chǔ)。 3、首次改進(jìn)了國(guó)外文獻(xiàn)中報(bào)道的單細(xì)胞5'-RACE獲取輕、重鏈可變區(qū)cDNA的方法,使用挑取單細(xì)胞克隆團(tuán)進(jìn)行5'-RACE,提高了效率的同時(shí)降低了操作的難度。 4、新型流感HA肽疫苗能夠誘導(dǎo)機(jī)體產(chǎn)生保護(hù)性抗體免疫應(yīng)答,對(duì)不同流感病毒亞型(H1N1和H5N1)具有交叉保護(hù)性。
[Abstract]:Influenza virus, as one of the most important human infectious viruses, has caused tremendous harm to human beings. It has caused three global influenza pandemics in the last century, causing tens of millions of deaths. Influence.
Although inactivated influenza vaccines have been developed to better control the persistent epidemic and increased virulence of the virus strain, little is known about the immunology and pathogenesis of the new influenza A (H1N1) virus. The research is of great significance.
In this study, we used baculovirus insect expression system to express a novel biological activity A H1N1 HA protein and NA protein. Based on this, we carried out the following two aspects of research:
Firstly, the preparation of human neutralizing monoclonal antibodies against a novel H1N1 HA protein was carried out. Memory B cells were transformed by EBV and transformed into B cells in the presence of CpG and feeder cells. Cell clones secreting specific antibodies against HA protein were screened after limited dilution. A total of 135 B cell samples containing 12960 strains were screened from 96U porous plates. Forty-five positive clonal cells were screened for many times. 36 of these positive clones died in the following 1-2 months, suggesting that most of the B cells transformed by EBV were short-lived, but only a few could survive for a long time (2 months). To avoid further loss of positive clonal cells, we compared the positive cells with K6H6/B5 cell lines. In the absence of immortal positive cells, this study changed the research strategy to obtain four pairs of positive cell light and heavy chain variable region cDNA genes by single cell clone 5'-RACE method, and then utilized baculovirus insects. The results showed that the insect cells were not suitable host cells for the expression of the genetic engineering antibody, although this study also had literature support in the use of insect expression.
Secondly, a novel HA epitope vaccine of influenza A virus was developed. Influenza viruses use continuous point mutation and gene recombination to induce continuous mutation and escape the memory immune response of the body. The design of epitope vaccines is usually based on linear epitopes, whether B cell epitopes or T cell epitopes. In this part, we designed peptide vaccines that mimic conformational epitopes and then immunized mice to evaluate whether they could induce antibody immune responses by using ELISA, hemagglutination inhibition test, neutralization test and peptide blocking ELISA. The results showed that the HA peptide vaccine immunized mice serum could bind peptide, recombinant H1N1 HA protein, recombinant H5N1 HA protein and inactivated H1N1 vaccine, inhibit hemagglutination test, and neutralize H1N1 and H5N1 viruses of different influenza virus subtypes. Fluid immune response and cross protection.
To sum up, this study has achieved the following main results:
1. Biologically active HA and NA proteins were successfully expressed by baculovirus expression system. Baculo Gold system and Bac-to-Bac system were compared. The results showed that the latter was more suitable for expressing HA protein.
2. EBV transformed B cells and screened 9 strains of B cells which could secrete HA-specific antibodies. Four pairs of light and heavy chain variable region cDNA genes of positive cells were obtained, which laid a foundation for further preparation of genetic engineering antibodies.
3. The method of obtaining light and heavy chain variable region cDNA from single cell 5'-RACE reported in foreign literatures was improved for the first time. The method of selecting single cell clone group for 5'-RACE was used to improve the efficiency and reduce the difficulty of operation.
4. The new influenza HA peptide vaccine can induce the body to produce protective antibody immune response, and has cross-protection against different influenza virus subtypes (H1N1 and H5N1).
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392.1
本文編號(hào)：2215641
[Abstract]:Influenza virus, as one of the most important human infectious viruses, has caused tremendous harm to human beings. It has caused three global influenza pandemics in the last century, causing tens of millions of deaths. Influence.
Although inactivated influenza vaccines have been developed to better control the persistent epidemic and increased virulence of the virus strain, little is known about the immunology and pathogenesis of the new influenza A (H1N1) virus. The research is of great significance.
In this study, we used baculovirus insect expression system to express a novel biological activity A H1N1 HA protein and NA protein. Based on this, we carried out the following two aspects of research:
Firstly, the preparation of human neutralizing monoclonal antibodies against a novel H1N1 HA protein was carried out. Memory B cells were transformed by EBV and transformed into B cells in the presence of CpG and feeder cells. Cell clones secreting specific antibodies against HA protein were screened after limited dilution. A total of 135 B cell samples containing 12960 strains were screened from 96U porous plates. Forty-five positive clonal cells were screened for many times. 36 of these positive clones died in the following 1-2 months, suggesting that most of the B cells transformed by EBV were short-lived, but only a few could survive for a long time (2 months). To avoid further loss of positive clonal cells, we compared the positive cells with K6H6/B5 cell lines. In the absence of immortal positive cells, this study changed the research strategy to obtain four pairs of positive cell light and heavy chain variable region cDNA genes by single cell clone 5'-RACE method, and then utilized baculovirus insects. The results showed that the insect cells were not suitable host cells for the expression of the genetic engineering antibody, although this study also had literature support in the use of insect expression.
Secondly, a novel HA epitope vaccine of influenza A virus was developed. Influenza viruses use continuous point mutation and gene recombination to induce continuous mutation and escape the memory immune response of the body. The design of epitope vaccines is usually based on linear epitopes, whether B cell epitopes or T cell epitopes. In this part, we designed peptide vaccines that mimic conformational epitopes and then immunized mice to evaluate whether they could induce antibody immune responses by using ELISA, hemagglutination inhibition test, neutralization test and peptide blocking ELISA. The results showed that the HA peptide vaccine immunized mice serum could bind peptide, recombinant H1N1 HA protein, recombinant H5N1 HA protein and inactivated H1N1 vaccine, inhibit hemagglutination test, and neutralize H1N1 and H5N1 viruses of different influenza virus subtypes. Fluid immune response and cross protection.
To sum up, this study has achieved the following main results:
1. Biologically active HA and NA proteins were successfully expressed by baculovirus expression system. Baculo Gold system and Bac-to-Bac system were compared. The results showed that the latter was more suitable for expressing HA protein.
2. EBV transformed B cells and screened 9 strains of B cells which could secrete HA-specific antibodies. Four pairs of light and heavy chain variable region cDNA genes of positive cells were obtained, which laid a foundation for further preparation of genetic engineering antibodies.
3. The method of obtaining light and heavy chain variable region cDNA from single cell 5'-RACE reported in foreign literatures was improved for the first time. The method of selecting single cell clone group for 5'-RACE was used to improve the efficiency and reduce the difficulty of operation.
4. The new influenza HA peptide vaccine can induce the body to produce protective antibody immune response, and has cross-protection against different influenza virus subtypes (H1N1 and H5N1).
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳慧;李崢;何小鵑;崔蓮仙;何維;;具有生物學(xué)活性的hMSH2蛋白在昆蟲(chóng)sf9細(xì)胞中的表達(dá)[J];基礎(chǔ)醫(yī)學(xué)與臨床;2008年07期
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