抗原性相同而免疫原性差异显著的两个蛋白对T、B细胞的激活作用研究

发布时间：2018-02-14 11:48

本文关键词： 戊型肝炎病毒颗粒性抗原疫苗 T细胞表位免疫原性抗原性　出处：《厦门大学》2008年硕士论文　论文类型：学位论文

【摘要】： 大肠杆菌表达的戊型肝炎病毒(HEV)衣壳蛋白ORF2的aa394-606片段NE2可以体外形成同源多聚体,其N端延伸突变体HEV 239蛋白(ORF2 aa368-606)在体外可以自发组装成类病毒颗粒。二者具有近乎完全一致的抗原性,但HEV 239的免疫原性比可溶的非颗粒蛋白NE2强200倍左右。有研究表明,颗粒蛋白抗原比可溶性抗原能够更加有效的被抗原递呈细胞捕获从而具有更强的免疫原性,亦有文献报道认为,这是由于颗粒抗原能更有效的激活B细胞。因此对HEV 239与NE2的研究将能使我们更好的了解颗粒抗原与可溶性抗原免疫原性差别产生的原因,并为将来合理的设计开发基因工程亚单位疫苗提供新的思路。本文第一部分研究了239蛋白与NE2蛋白诱导机体产生T细胞免疫应答的能力。用铝佐剂吸附的239蛋白与NE2蛋白皮下免疫BALB/c小鼠,免疫两周后取其脾细胞,用IL-5-ELISPOT与IFN-γ-ELISPOT方法来测定脾细胞中抗原特异性T细胞数量与类型。结果发现,239比NE2能激活更强的Th1与Th2应答,这可能是NE2蛋白免疫原性较弱的原因之一。本文第二部分利用覆盖HEV 239蛋白全长的15AA肽库(相邻肽段重叠10AA)做为IFN-γ-ELISPOT实验中的刺激原初步筛选HEV 239蛋白的H-2~d限制的T细胞表位。结果显示肽段P34(序列:HSKTFFVLPLRGKLS)有较强的反应性而P35(:序列FVLPLRGKLSFWEAG)有中等强度的反应,其他肽段无反应。对P34与P35肽中所含T细胞表型的进一步研究发现肽P34为优势Th表位肽而肽P35包含一较弱CTL表位和来自P34肽的部分Th表位活性。本文第三部分用IFN-γ-ELISPOT实验来定量分析不同浓度的239与NE2蛋白体外激活P34-CFA免疫小鼠脾细胞的能力。结果发现,高浓度的239与NE2都能激活P34特异的T细胞,而在低浓度抗原的情况下,239仍能诱导T细胞应答而NE2不能。实验结果说明,只有高浓度的NE2才能激活T细胞应答,而在疫苗免疫的过程中无法实现高的局部抗原浓度,因此NE2很难有效的激活T细胞应答。而为了验证激活Th细胞能力对于NE2蛋白免疫原性的影响,用P34,P35与P18(阴性对照)CFA初免小鼠后再腹腔加强5,10,20μg铝佐剂吸附的NE2蛋白,并对免疫后三周的小鼠血清中抗HEV抗体的进行检测,实验结果显示P34、P35均对NE2有初免效果,能帮助此剂量的NE2产生抗体应答,但P34的效果比P35强,而P18无初免效果。但是P34初免NE2加强的抗体反应仍弱于低剂量239蛋白诱导的抗体反应。这说明不能有效激活T细胞是NE2抗原性较弱的原因,但不是唯一原因。本研究的第四部分主要比较NE2与239蛋白活化B细胞能力的差异,将0.2,2,20,100μg铝佐剂吸附的HEV 239与NE2蛋白腹腔免疫Balb/c裸鼠,每组三只,检测免疫后第7天与第14天裸鼠血清中抗HEV抗体。结果发现仅0.2μgHEV 239就能诱导非T细胞依赖的抗体应答,而100μg的NE2蛋白仍无法诱导可检出的非T细胞依赖的抗体应答。这说明活化naive B细胞的能力不同也有可能是NE2与239具有相同的抗原性但不同免疫性的原因之一。最后,本文的第五部分主要将前面实验获得的H-2~d限制的Th表位应用于单克隆抗体的生产,对一无法利用基因工程技术表达的禽流感抗原,化学合成包含其B细胞表位与本研究获得的H-2~d限制的Th表位的肽段,将此肽段与CFA混合后免疫小鼠,最后我们获得了一株B表位特异的单克隆抗体。总之,本研究精确定位了HEV 239蛋白的H-2~d限制的Th表位与CTL表位,初步的阐明造成颗粒性抗原与可溶性抗原免疫原性差别的原因,为今后疫苗的研究开发提供了新的思路。最后,利用实验中获得的Th表位协助一B表位获得表位特异的单克隆抗体。
[Abstract]:Hepatitis E virus expressed in Escherichia coli (HEV) aa394-606 fragment of NE2 capsid protein ORF2 in vitro can form homo oligomers, the N mutant HEV protein 239 terminal extension (ORF2 aa368-606) in vitro can spontaneously assemble into virus like particles. The two have almost identical antigenicity of HEV 239, but the ratio of primary immune non soluble granule protein NE2 200 times. Studies have shown that particle protein antigen can be more effective than soluble antigen by antigen presenting cell capture immunogenicity which is more, also have reported in the literature that this is due to the particle antigen can effectively activate B cells. So the research on HEV 239 and NE2 will enable us to better understand granular antigen and soluble antigen immunogenicity difference, and provide new ideas for the design and development of genetic engineering subunit vaccine of reasonable future.
The first part of this research 239 protein and NE2 protein to induce T cell immune response. 239 protein and NE2 BALB/c mice were immunized subcutaneously with aluminum adjuvant adsorption, two weeks after immunization and the spleen cells, using IL-5-ELISPOT and IFN- gamma -ELISPOT method to determine the number and type of antigen specific T cells in spleen cells 239. The results showed that NE2 could activate Th1 and Th2 than the stronger response, which may be one of the reasons of weak immunogenicity of NE2 protein.
In the second part, by covering the full-length 15AA HEV 239 protein peptide library (adjacent peptides overlapping 10AA) as IFN- gamma in the -ELISPOT experiment to stimulate the primary screening of HEV 239 protein H-2~d restricted T cell epitopes. The results showed that the peptide P34 (sequence: HSKTFFVLPLRGKLS) with reactive P35 (strong: the sequence of FVLPLRGKLSFWEAG) had moderate reaction, other peptides without reaction. Further research on the phenotype of T cells containing P34 and P35 peptides found in peptide P34 was the dominant Th epitope peptide and peptide P35 contains a weak CTL epitope and P34 peptide from Th epitope.
The third part of the ability of IFN- gamma -ELISPOT experiment to quantitative analysis of different concentrations of P34-CFA 239 and NE2 protein in vitro activated immune spleen cells in mice. The results showed that high concentration of 239 and NE2 could activate P34 specific T cells, and in the low concentration of antigen, 239 can induce T cell response NE2 not only. The experimental results show that the high concentration of NE2 can activate T cell responses, and in the process of vaccine could not achieve high local concentration of antigen, so it is difficult for the activation of NE2 T cell responses. In order to verify the ability to activate Th cells to influence the immunogenicity of NE2 protein by P34 P35. With P18 (negative control) NE2 protein in mice after intraperitoneal free 5,10,20 g strengthened aluminium adjuvant adsorbed CFA, and anti HEV antibody in serum for three weeks after immunization in the detection, experimental results show that P34, P35 have a primary immune effect on NE2, can Help this dose of NE2 antibody response, but the effect of P34 is stronger than P35, while P18 had no effect. But the early free antibody response to P34 prime NE2 strengthening is still weaker than low dose 239 protein induced antibody response. It shows that you can't effectively activate NE2 is weak antigenicity of T cells, but it is not the only reason.
The fourth part of this study, the main difference between NE2 and 239 proteins of B cell activation, 0.2,2,20100 g aluminum adjuvant adsorbed HEV 239 and NE2 protein in nude mice by intraperitoneal immune Balb/c, three rats in each group, detected seventh days after immunization and fourteenth days of serum anti HEV antibody in nude mice. The results showed that the antibody response is only 0.2 ~ gHEV 239 can induce T cell dependent antibody response, and 100 g NE2 protein is not detectable by non T cell dependent. This shows that the ability of activated naive B cells may also be different is NE2 and 239 have the same antigenicity but different immunity is one of the reasons.
Finally, the fifth part of this paper will be in front of the experiments of H-2~d restricted Th epitopes used in the production of monoclonal antibodies against avian influenza A antigen, cannot be expressed by using genetic engineering technique, including chemical synthesis of B cell epitope peptide and the acquired H-2~d restricted Th epitopes, this peptide with the mixture of CFA mice. Finally we got a strain of monoclonal antibody B specific epitope.
In short, the accurate positioning of the Th HEV 239 protein and H-2~d restricted CTL epitopes, preliminary clarify the cause of granular antigen and soluble antigen immunogenicity difference, provides new ideas for the future development of vaccine research. Finally, using the obtained Th epitope to B epitope epitope specific monoclonal antibodies.

【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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