本文選題：A族鏈球菌(GAS)　切入點(diǎn)：Fba蛋白　出處：《河北醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的: A族乙型溶血性鏈球菌(group A streptococcus, GAS)是鏈球菌中致病性最強(qiáng)的一種,可長期寄居于人體鼻咽部和腸道中,引起各種化膿性炎癥、猩紅熱、丹毒、新生兒敗血癥、腦膜炎、產(chǎn)褥熱以及鏈球菌變態(tài)反應(yīng)性疾病等。臨床上存在著反復(fù)感染和難以治愈等問題。如果將菌體表面蛋白作為疫苗,將刺激機(jī)體產(chǎn)生相應(yīng)抗體,干擾鏈球菌侵入細(xì)胞,從而易于機(jī)體清除鏈球菌。候選的菌體蛋白中對M蛋白的了解最清楚,但由于M蛋白血清型太多,且M蛋白可能引起嚴(yán)重的變態(tài)反應(yīng),使得M蛋白作為疫苗的應(yīng)用受到限制。世界各地不同實(shí)驗(yàn)室的研究涉及到鏈球菌的F1蛋白、C5肽酶、鏈球菌糖、外毒素等,但免疫效果始終難以超越M蛋白。 Terao Y.等利用化膿性鏈球菌SF370網(wǎng)上公布的基因組序列,發(fā)現(xiàn)一新型菌體蛋白Fba,含有1068個核苷酸,編碼355個氨基酸,分子量為37.8Kda[1]。整個肽鏈從N端分為信號肽區(qū),雙螺旋結(jié)構(gòu)區(qū),富含脯氨酸區(qū)及C端的錨定區(qū)。它存在于13、22、28、44、49、60、67、75、77、79、80、82、87和89等多個血清型中,各型之間的Fba蛋白差異不大。實(shí)驗(yàn)結(jié)果顯示表達(dá)Fba蛋白的GAS(Fba+GAS)侵入上皮細(xì)胞的能力較強(qiáng)。它還可以與補(bǔ)體調(diào)節(jié)因子FH、FHL-1結(jié)合,以促進(jìn)鏈球菌黏附、入侵上皮細(xì)胞及抗吞噬作用[2],因此顯示它對鏈球菌在體內(nèi)的存活、毒力及致病性等諸多方面的重要作用[3]。本室前期研究發(fā)現(xiàn)Fba蛋白可誘發(fā)與M蛋白相當(dāng)?shù)谋Ｗo(hù)性免疫應(yīng)答,因此Fba蛋白具備作為GAS疫苗候選蛋白的極其有利的潛質(zhì)。 本研究在已知A族鏈球菌表面蛋白Fba具有良好的免疫原性和免疫保護(hù)性的基礎(chǔ)上,將分段克隆表達(dá)的Fba蛋白免疫動物并攻毒,以確定其具有保護(hù)性功能的區(qū)段。制備Fba蛋白的多抗,用純化的抗Fba蛋白的多抗篩選噬菌體隨機(jī)七肽庫(Ph·D-7),獲得多個Fba蛋白抗原的線性表位及構(gòu)象表位。 方法:1.1根據(jù)Fba蛋白的結(jié)構(gòu)特點(diǎn)將其劃分為四個重疊區(qū)域:37~110aa、68~161aa、104~277aa、160~324aa。分別命名為Fba1、Fba2、Fba3、Fba4。大量表達(dá)Fba分段蛋白,親和柱層析純化后免疫動物。 1.2將雌性Balb/c小鼠分為五組,每組8只,分別為FbaA1蛋白組、FbaA2蛋白組、FbaA3蛋白組、FbaA4蛋白組及PBS對照組,各組均以相同劑量蛋白免疫小鼠并加強(qiáng)免疫兩次,每次免疫均間隔兩周,并在每次免疫前內(nèi)眥取血,末次免疫后兩周取血。 1.3 ELISA檢測各免疫組血清IgG水平。使用致死量的GAS(M+Fba+)攻擊免疫三次后的小鼠,計算各組的保護(hù)率。 2.1將純化的Fba蛋白免疫新西蘭兔,以相同劑量蛋白免疫四次,每次免疫均間隔兩周,末次免疫后兩周頸動脈取血處死。ELISA檢測兔血清IgG水平。純化抗體,ELISA檢測純化后抗體水平。 2.2采用純化的IgG篩選噬菌體隨機(jī)七肽庫,經(jīng)3輪生物淘洗后,隨機(jī)挑取分隔良好的克隆,通過夾心ELISA試驗(yàn)檢測其與多抗的親和力,挑選其中親和力高的21個噬菌體克隆,提取單鏈DNA并測序。根據(jù)測序結(jié)果推導(dǎo)出其氨基酸序列,再與Fba蛋白序列進(jìn)行比較分析,推斷Fba蛋白的表位。 結(jié)果:1.1經(jīng)SDS-PAGE分析顯示成功表達(dá)并純化了Fba分段蛋白。 1.2將純化的Fba分段蛋白免疫動物,實(shí)驗(yàn)組血清IgG產(chǎn)生呈逐漸增高趨勢,其中以Fba2蛋白組效價升高最為明顯,達(dá)1:102400,依次為Fba3、Fba4及Fba1蛋白組。 1.3攻毒后5天,PBS組小鼠全部死亡,各實(shí)驗(yàn)組連續(xù)觀察兩周后依然有多只小鼠存活,各組保護(hù)率為Fba2蛋白組50%、Fba1、Fba3及Fba4蛋白組均為25%,經(jīng)SPSS統(tǒng)計分析各實(shí)驗(yàn)組與PBS組保護(hù)率有顯著差異。 2.1純化的Fba蛋白免疫新西蘭兔后獲得高IgG水平抗體。成功純化多抗。 2.2經(jīng)過三輪篩選后,獲得了高親和力的噬菌體克隆。對21株噬菌體克隆測序,推導(dǎo)出線性表位15個,構(gòu)象表位3個。 結(jié)論: 1 FbaA2段可誘導(dǎo)較強(qiáng)的保護(hù)性免疫應(yīng)答。 2獲得了多個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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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