新型甲型H1N1流感病毒血凝素蛋白人源化中和单抗和多肽表位疫苗研究
发布时间：2018-08-31 17:16
【摘要】：流感病毒作为最主要人类感染病毒之一,对人类的危害巨大。曾在上个世纪引起了三次全球性的流感大流行,造成数千万人的死亡。进入新世纪以来,在2009年爆发了全球性的流感大流行,给世界的社会与经济发展造成了一定程度的负面影响。 尽管已经研发出灭活流感疫苗,较好的控制了该病毒株的持续流行和毒性加剧。但是,目前针对新型甲型H1N1流感病毒的免疫学及其致病机理仍了解不多。血凝素(haemegglutinin, HA),作为最主要的参与病毒感染的表面抗原之一,对于H1N1病毒的研究具有重要意义。 本研究利用杆状病毒昆虫表达系统表达了具有生物学活性的新型甲型H1N1 HA蛋白和NA蛋白,以此为基础,开展了如下两方面研究： 首先,开展了新型甲型H1N1 HA蛋白人中和单克隆抗体的制备。利用EBV转化记忆性B细胞,在CpG和饲养细胞的存在下,培养转化B细胞；经有限稀释后筛选能分泌HA蛋白特异性抗体的细胞克隆,共筛选96U孔板135块包含12960株B细胞样品；最终,经多次筛选获得45株阳性克隆细胞。这些阳性细胞克隆中有36株阳性细胞克隆在随后1-2月中死亡,提示EBV转化的B细胞中绝大部分是短寿命的,而仅有少数才能长期存活(2月)。为避免阳性克隆细胞进一步丢失,我们将阳性细胞与K6H6/B5细胞系进行PEG融合以获得永生的阳性细胞。然而经多次实验,但却无法筛选到杂交瘤细胞。在不能获得永生阳性细胞的情况下,本研究改变研究策略,通过单细胞克隆团5'-RACE方法获得4对阳性细胞轻、重链可变区cDNA基因,进而利用杆状病毒昆虫细胞表达该基因工程抗体。结果表明,昆虫细胞不是基因工程抗体表达的合适宿主细胞,尽管本研究在采用昆虫表达时也有文献支持。下一步研究中需要使用更合适的CHO细胞表达上述HA特异性人基因工程抗体。 其次,本课题进行了新型甲型流感病毒HA表位肽疫苗研究。流感病毒利用连续点突变和基因重组导致不断变异,进而逃避机体的记忆免疫应答。目前的疫苗往往只是针对某一特定的病毒株或亚型,不能保护机体不被其他病毒株感染。而针对表位疫苗的设计,通常利用的是线性表位,无论是B细胞表位,还是T细胞表位。本部分课题通过设计能模拟构象表位的肽疫苗,进而免疫小鼠,利用ELISA、血凝抑制实验、中和实验以及肽阻断ELISA实验,评价其是否能够引起抗体免疫应答。结果显示,该HA肽疫苗免疫接种的小鼠血清能够结合肽、重组H1N1 HA蛋白、重组H5N1 HA蛋白以及H1N1灭活疫苗；能够抑制血凝实验,同时还能够中和非同种流感病毒亚型的H1N1和H5N1病毒。上述结果表明,新型HA肽疫苗能够诱导机体产生相应的体液免疫应答,并且具有交叉保护作用。 综上所述,本研究取得了以下主要成果： 1、利用杆状病毒表达系统成功表达出具有生物学活性的HA蛋白和NA蛋白,对BaculoGold system和Bac-to-Bac system这两种杆状病毒昆虫表达系统进行比较,结果表明后者更适合表达HA蛋白。 2、EBV转化B细胞,筛选到9株能够分泌HA特异性抗体的B细胞,进一步获得4对阳性细胞轻、重链可变区cDNA基因,为进一步制备基因工程抗体打下了基础。 3、首次改进了国外文献中报道的单细胞5'-RACE获取轻、重链可变区cDNA的方法,使用挑取单细胞克隆团进行5'-RACE,提高了效率的同时降低了操作的难度。 4、新型流感HA肽疫苗能够诱导机体产生保护性抗体免疫应答,对不同流感病毒亚型(H1N1和H5N1)具有交叉保护性。
[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).
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：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).
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1
【参考文献】
相关期刊论文 前1条
1 陈慧;李峥;何小鹃;崔莲仙;何维;;具有生物学活性的hMSH2蛋白在昆虫sf9细胞中的表达[J];基础医学与临床;2008年07期
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