α-1，6甘露糖转移酶基因缺陷的毕赤酵母菌在流感疫苗研究中的应用

发布时间：2018-07-05 12:33

本文选题：神经氨酸酶 + 糖基化　；参考：《中国人民解放军军事医学科学院》2009年硕士论文

【摘要】： 许多病毒包膜蛋白为糖蛋白,如流感病毒(Influenza Virus)的血凝素(Hemagglutinin, HA)和神经氨酸酶(Neuraminidase, NA),乙肝病毒(Hepatitis B Virus,HBV)的全长preS以及人免疫缺陷病毒(Human Immunodeficiency Virus,HIV)的gp120蛋白等。这些糖蛋白大多为病毒疫苗的主要保护性抗原。目前针对这些抗原的疫苗研究主要集中在对蛋白抗原表位或多糖抗原表位的研究上,而对糖基在糖蛋白疫苗中的作用研究较少。在许多抗原提呈细胞(Antigen Presenting Cells, APCs)上已发现大量的甘露糖受体,这提示甘露糖可能参与抗原的提呈。Lam等研究发现毕赤酵母(Pichia pastoris)表达的甘露糖基化的卵清白蛋白(Ovalbumin, OVA)比E.coli表达的非糖基化OVA具有更强的免疫原性[1]。从而提示甘露糖基化可能会增强抗原的免疫原性。现有的流感疫苗主要是鸡胚系统生产的,考虑到鸡胚作为疫苗生产系统时的耗时性等缺点,发展基因工程疫苗是其疫苗研究的一个重要方向。流感病毒的糖蛋白NA是流感疫苗的一个主要抗原成份,利用其来开发流感基因工程疫苗是流感疫苗研究的一个重要部分。酵母菌是一个良好的疫苗生产平台,已成功应用于HBV和人乳头瘤病毒(Human Papilloma Virus, HPV)等非糖蛋白疫苗的生产。但是,Martinet等研究发现,作为一个糖蛋白,流感病毒的N2型NA在野生型毕赤酵母菌GS115中表达时发生过度糖基化修饰,糖链上约含有30-40个甘露糖残基,这种糖基结构与天然病毒来源的NA的糖基结构差异很大,利用这种过度糖基化的NA作为疫苗免疫时,对鼠源性X47-流感病毒的致死性攻击只有50%的保护率,而且发现通过点突变缺失NA的部分糖基化位点以减少NA的糖含量,在一定程度上增强了NA抗原的免疫原性[2]。但是,这种点突变及糖基化位点的减少是否会导致NA在合成过程中的折叠错误进而引起的抗原表位的变化还有待研究。本研究在不改变NA的糖基化位点的前提下,通过阻断NA在酵母中的过度糖基化合成途径以降低NA的糖含量,进而研究糖基化程度改变对其免疫原性的影响。因此,本研究以α-1,6甘露糖转移酶(OCH1)基因缺陷的毕赤酵母菌(命名为GJK01)作为NA的表达菌株。OCH1基因是毕赤酵母菌过度甘露糖基化形成的一个关键起始酶基因,它的缺失阻断了酵母菌的过度甘露糖基化修饰,使糖基化修饰停留在低甘露糖基化水平。为了探索不同程度糖基化修饰与抗原免疫原性之间的关系,本研究选用了野生型毕赤酵母菌GS115、OCH1缺陷毕赤酵母菌GJK01以及大肠杆菌E.coli三种能进行不同程度糖基化修饰的表达系统作为研究平台。分别构建了酵母表达载体pPIC9-NA和大肠杆菌表达载体pET22b-NA,并转化野生型酵母菌GS115、OCH1缺陷酵母菌GJK01以及大肠杆菌E.coli BL21(DE3)。成功获得了含有NA基因的三种不同的重组菌株。通过重组菌株的培养,目的蛋白的纯化,最终纯化获得了三种不同宿主菌表达的重组蛋白NA。SDS-PAGE分析发现,GS115,GJK01和E.coli表达的NA的分子量分别为:75kDa,55kDa和45kDa,经N-糖肽特异性糖苷酶PNGaseF切割之后,上述各种NA的分子量均降低到45kDa,这与通过计算NA的氨基酸序列所得的理论值45 kDa是相一致的。因此,GS115表达的NA的糖含量为40%(高糖化),GJK01表达的NA的糖含量为18%(低糖化),E.coli表达的NA的糖含量为0%(非糖化)。将这三种糖基化程度不同的NA免疫BALB/c小鼠,并利用鸡胚生产的,包含N1型NA的商业化流行性感冒病毒裂解疫苗作为ELISA包被抗原,来检测不同NA诱导小鼠产生的针对天然NA的抗体滴度。结果显示,1μg/只小鼠剂量免疫时,二次免疫之后,只有低糖化NA能诱导较高抗体滴度的产生,其滴度达到1:5500,而高糖化与非糖化NA几乎不能诱导抗体的产生,其滴度分别仅为:1:10 (P0.01)和1:13 (P0.01)。三次免疫之后,高糖化NA诱导产生的抗体滴度仍然很低,只有1:20 (P0.05),非糖化NA诱导产生抗体滴度稍微有所增加,达到1:630。低糖化NA三次免疫后诱导产生的抗体水平与二次免疫之后的水平相当,滴度为1:4700。以此为基础,又进一步研究了用于免疫的NA抗原的剂量与其诱导产生的特异性抗体滴度之间的关系。结果显示,二次免疫之后,随着剂量由0.2μg增加到1μg,低糖化NA诱导产生的抗体滴度从1:70增加到1:5500;而高糖化NA诱导产生的抗体滴度仅为1:20和1:10,进一步提高剂量到3μg时,高糖化NA才能诱导产生1:6500的抗体滴度。非糖化NA组诱导产生抗体滴度的变化趋势与对应剂量的高糖化NA组相似。三次免疫之后,0.2μg剂量时,低糖化NA能诱导1:4900抗体滴度的产生,而高糖化NA诱导产生的抗体滴度值只为1:10 (P0.01)。非糖化NA组诱导产生的抗体滴度的变化趋势与对应剂量的高糖化NA组相似,都只有在剂量增加到3μg时,才能诱导高的抗体滴度的产生。当剂量达到3μg时,三种NA均能诱导较高抗体滴度的产生,分别为:高糖化NA组为1:36000,低糖化NA组为1:25000,非糖化NA组为1:7000。以上结果显示,不同于野生型毕赤酵母菌GS115表达的过度糖基化的NA,OCH1缺陷的毕赤酵母菌GJK01表达的NA为低糖基化修饰的糖蛋白,而且相比于高糖化或非糖化NA,该低糖化NA具有更强的诱导小鼠产生针对天然NA的特异性抗体的能力。这提示了低糖化NA有可能成为候选的基因工程流感疫苗,具有低糖化修饰的OCH1缺陷毕赤酵母菌GJK01有望成为一种新型的糖蛋白疫苗研究和开发平台。
[Abstract]:Many virus envelope proteins are glycoproteins such as Influenza Virus Hemagglutinin (HA) and neuraminidase (Neuraminidase, NA), the full length preS of the hepatitis B virus (Hepatitis B Virus, HBV), and the human immunodeficiency virus (Human). The major protective antigens of the vaccine are currently focused on the study of protein epitopes or polysaccharides epitopes, but less research on the role of glycosyl in glycoprotein vaccines. A large number of mannose receptors have been found on Antigen Presenting Cells (APCs), which is a hint. Mannose may participate in antigen presentation.Lam and other studies found that the mannose based glycosylated ovalbumin (Ovalbumin, OVA) expressed in Pichia pastoris (Pichia pastoris) has a stronger immunogenic [1]. than the non glycosylated OVA expressed in E.coli, suggesting that mannose may enhance the immunogenicity of the antigen.
The existing influenza vaccine is mainly produced by the chicken embryo system. Considering the disadvantages of the chicken embryo as the time consuming of the vaccine production system, the development of the gene engineering vaccine is an important direction of its vaccine research. The glycoprotein NA of influenza virus is a major antigen component of the influenza vaccine. An important part of the vaccine research, yeast is a good vaccine production platform and has been successfully used in the production of HBV and Human Papilloma Virus (HPV) and other non glycoprotein vaccines. However, Martinet and other studies have found that as a glycoprotein, the N2 NA of the flu virus is in the GS115 form of wild Pichia pastoris. There are about 30-40 mannose residues in the sugar chain, which differs greatly from the glycosyl structure of NA from the natural virus. When the NA is used as a vaccine, the lethal attack of the mouse derived X47- influenza virus is only 50%, and the point mutation is found through the point mutation. A partial glycosylation site missing NA to reduce the sugar content of NA, to a certain extent, enhanced the immunogenicity of the NA antigen, but whether this point mutation and the reduction of glycosylation sites will lead to the changes in the epitopes caused by the folding errors of NA in the synthesis process still remain to be studied. This study does not change the glycosylation sites of NA. On the premise of point, by blocking the excessive glycosylation pathway of NA in yeast to reduce the sugar content of NA, and then study the effect of the change of glycosylation on its immunogenicity. Therefore, the.OCH1 gene of NA, the expression strain of NA, was used as the expression strain of the strain of -1,6 mannose transferase (OCH1) gene (named GJK01) as the.OCH1 gene. A key starting enzyme gene formed by the excessive mannose formation of the mother bacteria, the deletion of which blocks the excessive mannose modification of yeast and makes the glycosylated modification stay at the level of low mannose.
In order to explore the relationship between glycosylation and immunogenicity of different degrees of glycosylation, the study selected the wild type Pichia pastoris GS115, OCH1 defective Pichia GJK01 and Escherichia coli E.coli for three different degrees of glycosylated expression system as the research platform.
Yeast expression vector pPIC9-NA and Escherichia coli expression vector pET22b-NA were constructed respectively, and wild yeast GS115, OCH1 deficient yeast GJK01 and Escherichia coli E.coli BL21 (DE3) were transformed. Three different recombinant strains containing NA gene were successfully obtained.
Through the culture of the recombinant strain and purification of the target protein, the recombinant protein NA.SDS-PAGE analysis of three different host bacteria was obtained. The molecular weight of NA expressed in GS115, GJK01 and E.coli were 75kDa, 55kDa and 45kDa respectively. After the N- glycoseptide specific glycosidase PNGaseF was cut, the above NA molecular weight was reduced to 45. KDa, which is the same as the theoretical value 45 kDa obtained by the amino acid sequence calculated by NA. Therefore, the sugar content of NA expressed by GS115 is 40% (Gao Tanghua), the content of the sugar content of NA expressed by GJK01 is 18% (low saccharification), and the content of NA in E.coli is 0% (non saccharification).
The three NA BALB/c mice were immunized with different glycosylation levels, and the commercial Inactivated Split Influenza Vaccine containing N1 NA was used as a ELISA package to detect the antibody titers of natural NA induced by different NA induced mice.
The results showed that only low saccharification NA could induce the production of high antibody titer only after two doses of immunization at 1 g/ mice, and its titer reached 1:5500, while high saccharification and non saccharification NA could hardly induce antibody production, and its titer was only 1:10 (P0.01) and 1:13 (P0.01). After three immunizations, the antibody induced by high saccharification NA was induced. The titer was still very low, only 1:20 (P0.05), the titer of the antibody induced by non saccharification NA increased slightly, and the level of antibody induced by the three immunization of 1:630. low saccharification NA was equivalent to the level after the immunization, the titer was 1:4700.
On this basis, we further studied the relationship between the dose of NA antigen used for immunization and the specific antibody titer induced by immunization.
The results showed that the antibody titer induced by low saccharification NA increased from 1:70 to 1:5500 as the dose increased from 0.2 g to 1 mu g, and the titer of antibody induced by high saccharification NA was only 1:20 and 1:10, and the high glucose NA could induce the antibody titer to produce 1:6500 when the dose to 3 mu G was further increased. Non saccharifying NA group induced resistance to produce resistance. The trend of body titer is similar to that of the high glycated NA group.
After three immunization, low saccharification NA can induce the production of 1:4900 antibody titer at 0.2 g dose, while the titer of antibody produced by high saccharification NA is only 1:10 (P0.01). The trend of antibody titer induced by non saccharifying NA group is similar to that of the high saccharification NA group corresponding to the corresponding dose, only when the dose increases to 3 u g, the high resistance can be induced. When the dose reached 3 G, three kinds of NA could induce high antibody titer, respectively: high glucose NA group was 1:36000, low saccharification NA group was 1:25000, non saccharifying NA group was 1:7000.
The above results show that, unlike the over glycosylated NA of the wild type Pichia pastoris GS115, the NA of OCH1 deficient Pichia GJK01 expressed as a low glycosylated glycoprotein, and compared to the high saccharification or non saccharifying NA, the low saccharifying NA has a stronger ability to induce mice to produce specific antibodies against natural NA. It is shown that low saccharification NA may become a candidate gene engineering influenza vaccine. The low saccharification modified OCH1 deficient Pichia GJK01 is expected to become a new research and development platform for glycoprotein vaccine.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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