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)布時間：2018-08-31 17:16
【摘要】：流感病毒作為最主要人類感染病毒之一,對人類的危害巨大。曾在上個世紀引起了三次全球性的流感大流行,造成數(shù)千萬人的死亡。進入新世紀以來,在2009年爆發(fā)了全球性的流感大流行,給世界的社會與經(jīng)濟發(fā)展造成了一定程度的負面影響。 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,目前針對新型甲型H1N1流感病毒的免疫學(xué)及其致病機理仍了解不多。血凝素(haemegglutinin, HA),作為最主要的參與病毒感染的表面抗原之一,對于H1N1病毒的研究具有重要意義。 本研究利用桿狀病毒昆蟲表達系統(tǒng)表達了具有生物學(xué)活性的新型甲型H1N1 HA蛋白和NA蛋白,以此為基礎(chǔ),開展了如下兩方面研究： 首先,開展了新型甲型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細胞,在CpG和飼養(yǎng)細胞的存在下,培養(yǎng)轉(zhuǎn)化B細胞；經(jīng)有限稀釋后篩選能分泌HA蛋白特異性抗體的細胞克隆,共篩選96U孔板135塊包含12960株B細胞樣品；最終,經(jīng)多次篩選獲得45株陽性克隆細胞。這些陽性細胞克隆中有36株陽性細胞克隆在隨后1-2月中死亡,提示EBV轉(zhuǎn)化的B細胞中絕大部分是短壽命的,而僅有少數(shù)才能長期存活(2月)。為避免陽性克隆細胞進一步丟失,我們將陽性細胞與K6H6/B5細胞系進行PEG融合以獲得永生的陽性細胞。然而經(jīng)多次實驗,但卻無法篩選到雜交瘤細胞。在不能獲得永生陽性細胞的情況下,本研究改變研究策略,通過單細胞克隆團5'-RACE方法獲得4對陽性細胞輕、重鏈可變區(qū)cDNA基因,進而利用桿狀病毒昆蟲細胞表達該基因工程抗體。結(jié)果表明,昆蟲細胞不是基因工程抗體表達的合適宿主細胞,盡管本研究在采用昆蟲表達時也有文獻支持。下一步研究中需要使用更合適的CHO細胞表達上述HA特異性人基因工程抗體。 其次,本課題進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)點突變和基因重組導(dǎo)致不斷變異,進而逃避機體的記憶免疫應(yīng)答。目前的疫苗往往只是針對某一特定的病毒株或亞型,不能保護機體不被其他病毒株感染。而針對表位疫苗的設(shè)計,通常利用的是線性表位,無論是B細胞表位,還是T細胞表位。本部分課題通過設(shè)計能模擬構(gòu)象表位的肽疫苗,進而免疫小鼠,利用ELISA、血凝抑制實驗、中和實驗以及肽阻斷ELISA實驗,評價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；能夠抑制血凝實驗,同時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)機體產(chǎn)生相應(yīng)的體液免疫應(yīng)答,并且具有交叉保護作用。 綜上所述,本研究取得了以下主要成果： 1、利用桿狀病毒表達系統(tǒng)成功表達出具有生物學(xué)活性的HA蛋白和NA蛋白,對BaculoGold system和Bac-to-Bac system這兩種桿狀病毒昆蟲表達系統(tǒng)進行比較,結(jié)果表明后者更適合表達HA蛋白。 2、EBV轉(zhuǎn)化B細胞,篩選到9株能夠分泌HA特異性抗體的B細胞,進一步獲得4對陽性細胞輕、重鏈可變區(qū)cDNA基因,為進一步制備基因工程抗體打下了基礎(chǔ)。 3、首次改進了國外文獻中報道的單細胞5'-RACE獲取輕、重鏈可變區(qū)cDNA的方法,使用挑取單細胞克隆團進行5'-RACE,提高了效率的同時降低了操作的難度。 4、新型流感HA肽疫苗能夠誘導(dǎo)機體產(chǎn)生保護性抗體免疫應(yīng)答,對不同流感病毒亞型(H1N1和H5N1)具有交叉保護性。
[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)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392.1
本文編號：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)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392.1
【參考文獻】
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sf9細胞中的表達[J];基礎(chǔ)醫(yī)學(xué)與臨床;2008年07期
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