GAS表面蛋白Fba保护性区段的鉴定及其表位的筛选

发布时间：2018-03-24 18:50

本文选题：A族链球菌(GAS)　切入点：Fba蛋白　出处：《河北医科大学》2009年硕士论文

【摘要】： 目的: A族乙型溶血性链球菌(group A streptococcus, GAS)是链球菌中致病性最强的一种,可长期寄居于人体鼻咽部和肠道中,引起各种化脓性炎症、猩红热、丹毒、新生儿败血症、脑膜炎、产褥热以及链球菌变态反应性疾病等。临床上存在着反复感染和难以治愈等问题。如果将菌体表面蛋白作为疫苗,将刺激机体产生相应抗体,干扰链球菌侵入细胞,从而易于机体清除链球菌。候选的菌体蛋白中对M蛋白的了解最清楚,但由于M蛋白血清型太多,且M蛋白可能引起严重的变态反应,使得M蛋白作为疫苗的应用受到限制。世界各地不同实验室的研究涉及到链球菌的F1蛋白、C5肽酶、链球菌糖、外毒素等,但免疫效果始终难以超越M蛋白。 Terao Y.等利用化脓性链球菌SF370网上公布的基因组序列,发现一新型菌体蛋白Fba,含有1068个核苷酸,编码355个氨基酸,分子量为37.8Kda[1]。整个肽链从N端分为信号肽区,双螺旋结构区,富含脯氨酸区及C端的锚定区。它存在于13、22、28、44、49、60、67、75、77、79、80、82、87和89等多个血清型中,各型之间的Fba蛋白差异不大。实验结果显示表达Fba蛋白的GAS(Fba+GAS)侵入上皮细胞的能力较强。它还可以与补体调节因子FH、FHL-1结合,以促进链球菌黏附、入侵上皮细胞及抗吞噬作用[2],因此显示它对链球菌在体内的存活、毒力及致病性等诸多方面的重要作用[3]。本室前期研究发现Fba蛋白可诱发与M蛋白相当的保护性免疫应答,因此Fba蛋白具备作为GAS疫苗候选蛋白的极其有利的潜质。本研究在已知A族链球菌表面蛋白Fba具有良好的免疫原性和免疫保护性的基础上,将分段克隆表达的Fba蛋白免疫动物并攻毒,以确定其具有保护性功能的区段。制备Fba蛋白的多抗,用纯化的抗Fba蛋白的多抗筛选噬菌体随机七肽库(Ph·D-7),获得多个Fba蛋白抗原的线性表位及构象表位。方法:1.1根据Fba蛋白的结构特点将其划分为四个重叠区域:37~110aa、68~161aa、104~277aa、160~324aa。分别命名为Fba1、Fba2、Fba3、Fba4。大量表达Fba分段蛋白,亲和柱层析纯化后免疫动物。 1.2将雌性Balb/c小鼠分为五组,每组8只,分别为FbaA1蛋白组、FbaA2蛋白组、FbaA3蛋白组、FbaA4蛋白组及PBS对照组,各组均以相同剂量蛋白免疫小鼠并加强免疫两次,每次免疫均间隔两周,并在每次免疫前内眦取血,末次免疫后两周取血。 1.3 ELISA检测各免疫组血清IgG水平。使用致死量的GAS(M+Fba+)攻击免疫三次后的小鼠,计算各组的保护率。 2.1将纯化的Fba蛋白免疫新西兰兔,以相同剂量蛋白免疫四次,每次免疫均间隔两周,末次免疫后两周颈动脉取血处死。ELISA检测兔血清IgG水平。纯化抗体,ELISA检测纯化后抗体水平。 2.2采用纯化的IgG筛选噬菌体随机七肽库,经3轮生物淘洗后,随机挑取分隔良好的克隆,通过夹心ELISA试验检测其与多抗的亲和力,挑选其中亲和力高的21个噬菌体克隆,提取单链DNA并测序。根据测序结果推导出其氨基酸序列,再与Fba蛋白序列进行比较分析,推断Fba蛋白的表位。结果:1.1经SDS-PAGE分析显示成功表达并纯化了Fba分段蛋白。 1.2将纯化的Fba分段蛋白免疫动物,实验组血清IgG产生呈逐渐增高趋势,其中以Fba2蛋白组效价升高最为明显,达1:102400,依次为Fba3、Fba4及Fba1蛋白组。 1.3攻毒后5天,PBS组小鼠全部死亡,各实验组连续观察两周后依然有多只小鼠存活,各组保护率为Fba2蛋白组50%、Fba1、Fba3及Fba4蛋白组均为25%,经SPSS统计分析各实验组与PBS组保护率有显著差异。 2.1纯化的Fba蛋白免疫新西兰兔后获得高IgG水平抗体。成功纯化多抗。 2.2经过三轮筛选后,获得了高亲和力的噬菌体克隆。对21株噬菌体克隆测序,推导出线性表位15个,构象表位3个。结论: 1 FbaA2段可诱导较强的保护性免疫应答。 2获得了多个Fba蛋白抗原表位。
[Abstract]:Objective: group A hemolytic streptococcus (group A, Streptococcus, GAS) is a kind of Pathogenic Streptococcus, long-term living in human nasopharynx and the intestinal tract, cause purulent inflammation, fever, erysipelas, neonatal sepsis, meningitis, puerperal fever streptococcus and allergic diseases in clinic. The problem of repeated infection and difficult to cure. If the cell surface protein as a vaccine, will stimulate the body to produce antibodies, interference of Streptococcus invade cells, which is easy to remove the body of Streptococcus. The candidate mycelium protein of M protein is best understood, but because of too many serotypes of M protein and M protein may cause allergy. The reaction is serious, the M protein as a vaccine application is limited by different laboratories around the world. The research involves the streptococcal F1 protein, C5 peptidase, Streptococcus exotoxin, but without sugar The effect of pestilence is always difficult to surpass M protein.
Terao Y. using the genomic sequence of Streptococcus pyogenes SF370 published online, found a new protein Fba, containing 1068 nucleotides, encoding 355 amino acids, the molecular weight of the peptide 37.8Kda[1]. from N terminal is divided into signal peptide, double helix region, proline rich region and C end of the anchor area. It exists 13,22,28,44,49,60,67,75,77,79,80,82,87 and 89 other serotypes, Fba protein differences between various types of small. Experimental results show that the expression of Fba GAS (Fba+GAS) invasion of epithelial cells. It also can be used with the regulation of complement factor FH, FHL-1 binding, in order to promote the adhesion of Streptococcus, invasion of epithelial cells and the anti phagocytic effect of [2] therefore, it shows the in vivo survival of Streptococcus, an important role in many aspects of virulence and pathogenicity of [3]. in our previous study found that Fba protein and M protein can induce a protective immunity In response, the Fba protein has an extremely favorable potential as a candidate for the GAS vaccine.
This study has known in group A streptococcal Fba good immunogenicity and protective immunity on the basis of the piecewise Fba protein in animal cloning expression and challenged to determine the protective function of the section. The preparation of Fba protein antibodies, phage random seven peptide library with purified anti Fba protein polyclonal antibody (Ph - D-7), multiple Fba protein antigen epitopes and mimotopes.
Methods: 1.1. According to the structural characteristics of Fba protein, it was divided into four overlapping regions: 37~110aa, 68~161aa, 104~277aa and 160~324aa., respectively named Fba1, Fba2, Fba3, Fba4., a large number of Fba segment proteins, and purified by affinity column chromatography.
1.2 female Balb/c mice were divided into five groups, 8 rats in each group, respectively FbaA1 group, FbaA2 group, FbaA3 group, FbaA4 protein group and PBS control group, each group with the same dose of immunized mice and strengthen the immune two times, each time interval of two weeks were immune, and in every immunity before canthus blood, blood samples were collected two weeks after the last immunization.
The serum levels of IgG were detected by 1.3 ELISA. The mice were attacked after three times of immunization with lethal dose of GAS (M+Fba+), and the protection rate of each group was calculated.
2.1, the purified Fba protein was used to immunize New Zealand rabbits. The same dose of protein was immunized for four times. Each immunization interval was two weeks. After the last two weeks of the last immunization, the blood was collected from the carotid artery..ELISA was used to detect the serum IgG level. Purified antibody and ELISA were used to detect the antibody level after purification.
2.2 using the purified IgG phage random seven peptide library. After 3 rounds of biopanning, were randomly selected and separated good clones with PAA affinity was detected by sandwich ELISA test, selected 21 phage clones with high affinity, extraction of single stranded DNA and sequenced. The sequencing result according to its amino acid sequence was deduced, then compared with the Fba protein sequence, deduced Fba protein epitope.
Results: 1.1 SDS-PAGE analysis showed that Fba segmented protein was successfully expressed and purified.
1.2, purified Fba fragment protein was used to immunize animals. The serum IgG level of experimental group increased gradually, and the titer of Fba2 protein group increased most significantly, reaching 1:102400, followed by Fba3, Fba4 and Fba1 protein group.
5 days after the 1.3 attack, all the mice in the PBS group died. After two weeks of continuous observation, the mice in each group survived. The protection rate of each group was Fba2 protein group 50%, while the Fba1, Fba3 and Fba4 protein groups were all 25%. After statistical analysis by SPSS, the protection rates of the experimental group and the PBS group were significantly different.
2.1 New Zealand rabbits were immunized with purified Fba protein to obtain the high level of IgG. The successful purification of polyclonal antibody.
2.2 after three rounds of screening, high affinity phage clones were obtained. 21 phage clones were sequenced, and 15 linear epitopes were derived and 3 conformation epitopes were derived.
Conclusion:
The 1 FbaA2 segment could induce a strong protective immune response.
2 a number of Fba protein epitopes were obtained.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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