幽门螺杆菌外膜蛋白Omp18与UreB414融合疫苗的实验研究

发布时间：2018-02-27 01:35

本文关键词： 幽门螺杆菌外膜蛋白 OMP18 免疫保护粘膜免疫　出处：《第三军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的: 幽门螺杆菌(H.pylori)的全球感染率超过了50%,它是慢性活动性胃炎和消化性溃疡的主要病因,与肠型胃癌和胃黏膜相关淋巴样组织(MALT)淋巴瘤的发生密切相关。现有的根除H.pylori的手段主要是联合应用抗生素,但由于该菌感染的普遍性、耐药菌株的不断增加、较高的花费以及病人的低依从性等,限制了抗菌药物的疗效。因此,接种简便、成本低廉并易于大面积推广的H.pylori疫苗的研究十分迫切。在疫苗研究中,筛选有效的免疫抗原是关键性的环节。目前大多数疫苗采用的是与Hp致病相关的毒力因子作为抗原成分,如尿素酶(urease)、热休克蛋白(heat shockprotein,Hsp)、空泡毒素(vacuolating cytotoxin,VacA)、细胞毒素相关抗原(cytotoxinassociate antigen,CagA)、中性粒细胞激活蛋白(neutrophil-activating protein,NAP)等。革兰氏阴性细菌外膜蛋白具有良好的抗原活性,这些外膜蛋白作为抗体和免疫细胞攻击的主要靶标,可以介导对细菌最直接有效的杀灭作用,是决定免疫反应是否对人体具有保护性的关键因素。OMP18和UerB都是Hp的外膜蛋白成分,其编码基因具有高度保守性,也是Hp疫苗重要的免疫抗原。但一直令学者们遗憾的是每种抗原单独免疫动物时,获得的保护率均不是很高,而多种抗原联合免疫才能达到较好的效果。因此,本研究拟对上述二种保护性抗原基因进行克隆表达,并与分子内佐剂LTB串联得到多亚单位的融合蛋白,分别观察在小鼠体内诱导的不同免疫应答和保护作用,为筛选有效的H.pylori疫苗抗原奠定基础。方法: 1、采用PCR方法从H.pylori临床分离株9806基因组中扩增OMP18基因片段并构建在原核表达载体pQE-30中,通过酶切、测序鉴定阳性重组子。应用生物信息学软件DNAssist和GenBank数据库对已公布的H.pylori OMP18基因序列进行相似性分析。将含目的基因片段的重组载体转化大肠杆菌M15中,经IPTG诱导表达,用AKTA-explore纯化仪纯化表达的重组蛋白rOMP18。应用Tris-Tricine方法对表达产物和纯化产物进行分析,用纯化的rOMP18免疫家兔,并采用Western blot和免疫扩散试验鉴定重组蛋白的免疫性。 2、采用重叠延伸PCR的方法,分别以UreB414活性片段做为融合蛋白前端,与OMP18及粘膜佐剂LTB依次串联,和以粘膜佐剂LTB做为融合蛋白前端与OMP18依次串联,在片段间引入5个氨基酸的linker PQDPP进行连接;将融合基因构建在原核表达载体pET-28a中,经酶切及测序鉴定后,阳性重组子转化宿主菌E.coliBL21(DE3),IPTG诱导重组工程菌表达。Tris-Tricine电泳和免疫印迹鉴定融合蛋白,DNAssist软件分析融合蛋白理化特性,通过纯化条件摸索,AKTA-explore纯化仪纯化融合蛋白LTB-OMP18(LO)和UreB414-OMP18-LTB(UOL)。纯化后的融合蛋白分别与GM1神经节苷脂进行结合试验。 3、将144只小鼠随机分为6组:PBS对照组、单亚单位分子内佐剂融合蛋白组(UreB414-LTB)、双亚单位分子内佐剂融合蛋白组(UreB414-OMP18-LTB、)单一亚单位分子内佐剂融合蛋白组(LTB-OMP18)、体外物理混合组(rOMP18+UreB414-LTB)无分子内佐剂单一亚单位蛋白组(OMP18)。分别于0、7、14、28天口服免疫,剂量为150μg/只/次。术次免疫后10天,每组取12只小鼠剖杀取样,ELISA检测口服免疫后血清特异性IgG、IgA,粪便、肠道冲洗液sIgA水平。每组余下12只口服10~8 CFU Hp小鼠适应株,30天后,取小鼠胃部标本通过Hp培养及特异性PCR检测,确定Hp感染率,并计算口服免疫后各组攻毒保护率。结果: 1、经酶切鉴定和基因测序结果显示,H.pylori OMP18基因被正确克隆到pQE-30中。OMP18基因的核苷酸序列长度为540bp,与GenBank中序列比较,同源性为99%,氨基酸序列的同源性为100%。重组工程菌IPTG诱导,经Tris-Tricine分析,表达的目的蛋白分子量约为20KD,其表达产物占菌体蛋白的20%,为包涵体形式表达,经AKTA-explore 100蛋白纯化系统纯化,纯化后得到纯度＞85%的目的蛋白。以纯化蛋白为抗原加弗氏佐剂免疫家兔,制备兔抗rOMP18特异抗体,经免疫双扩散法检测,兔抗血清抗体效价为1:32,Western Blot结果也显示重组蛋白能被兔抗H.pylori血清所识别。 2、经酶切鉴定和基因测序结果显示,成功构建了融合基因LTB-OMP18和UreB414-OMP18-LTB,将融合基因LO和UOL克隆到pET28a载体上,得到融合蛋白表达载体pLO和pUOL,DnaStra软件评价蛋白linker具有较好的柔韧性。将两个阳性重组子转化至E.coli BL21(DE3)后,37℃培养经IPTG诱导,表达的融合蛋白经SDS-PAGE和免疫印迹分析显示分子量约为31KD和43KD。UVP扫描证实融合蛋白表达约占总蛋白的25%和20%。包涵体鉴定试验证实融合蛋白主要以包涵体形式表达,确定了采用亲和层析的纯化方法。纯化后得到纯度＞85%的融合蛋白。纯化后的两个融合蛋白分别能与GM1神经节苷脂发生结合反应,实验证实重组融合蛋白中LTB组分具有与GM1神经节苷脂结合的生物活性,说明融合蛋白保持了LTB的天然活性,具有粘膜佐剂活性。 3、ELISA检测融合蛋白rLO和rUOL免疫小鼠后血清特异性IgG、IgA,粪便、肠道冲洗液sIgA水平与PBS组相比,升高明显,差异极显著(p＜0.001),与亚单位免疫组相比,差异显著(p＜0.05),与多亚单位蛋白物理混合组相比,差异不显著(p＞0.05)。免疫攻毒后,免疫保护率为:rLO组67%(8/12),rUOL组83%(10/12)。统计分析显示,多亚单位融合蛋白组免疫保护率与PBS组相比,差异极显著(p＜0.001),与rOMP18组相比,差异显著(p＜0.05),与rOMP18+UreB414-LTB组相比,差异不显差(p＞0.05)。结论: 1、成功克隆了H.pylori OMP18基因,与GenBank已公布的H.pylori菌株基因序列具有高度同源性。纯化的重组蛋白rOMP18,具有良好的免疫原性和免疫反应性,可以作为H.pylori基因工程疫苗候选抗原。 2、成功构建、表达融合蛋白rLO和rUOL,纯化后的融合蛋白保持各亚单位组分及佐剂的独立免疫反应性,融合基因间的Linker对融合蛋白的立体构象影响较小。 3、小鼠体内特异性抗体水平及免疫保护效果都证明融合蛋白rLO和rUOL具有良好的安全性和免疫保护效果。
[Abstract]:Objective:
Helicobacter pylori (H.pylori) infection rate of more than 50% of the world, it is a major cause of chronic gastritis and peptic ulcer, and intestinal type of gastric cancer and gastric mucosa associated lymphoid tissue lymphoma (MALT) is closely related to the occurrence of. Existing H.pylori eradication is the main means of combined application of antibiotics, but because of its universality bacterial infection, increasing resistant strains, high cost, low patient compliance, limiting the efficacy of antibacterial agents. Therefore, vaccination is simple, low cost and easy to study large-scale promotion of the H.pylori vaccine is very urgent.
In vaccine research, screening the immune antigen effectively is a key link. At present most vaccines used is associated with Hp pathogenic virulence factors as antigen components, such as urease (urease), heat shock proteins (heat, shockprotein, Hsp) (vacuolating cytotoxin, VacA VacA), cytotoxin associated antigen (cytotoxinassociate antigen, CagA), neutrophil activating protein (neutrophil-activating, protein, NAP). The gram negative bacterial outer membrane protein has good antigenicity, the outer membrane protein as the main target of antibodies and immune cells attack, can mediate the killing effect on bacteria is the most direct and effective, determine whether the immune response is the key factor of protection the.OMP18 and UerB on the human body is the outer membrane protein component of Hp, its encoding gene is highly conservative, but also an important immune antigen Hp vaccine but has. It is regrettable that when each antigen is immune to animals, the protection rate is not very high, and the combined immunization of various antigens can achieve better results.
Therefore, this study intends to clone the expression of the two kinds of protective antigen gene, and in series with the intra molecular adjuvant LTB subunit fusion protein were observed in different immune response and protective effect in mice induced by H.pylori, to lay the foundation for the screening of effective vaccine antigen.
Method:
1, PCR method was used to isolate 9806 amplified OMP18 gene fragment and constructed into prokaryotic expression vector pQE-30 H.pylori from clinical, by enzyme digestion, sequencing of positive recombinants. Using bioinformatics software DNAssist and GenBank database on H.pylori OMP18 based similarity analysis published by sequence. The recombinant vector containing the target gene was transformed into Escherichia coli M15, expressed by IPTG induction, the recombinant protein was purified with AKTA-explore rOMP18. using Tris-Tricine method for expression and purification of expressed products were analyzed and purified, rOMP18 was used to immunize rabbits with purified, and the immunity Western blot and immunodiffusion test identification of recombinant protein.
2, using overlap extension PCR method, respectively with the active fragment of UreB414 fusion protein as a front end, and OMP18 and LTB are connected in series and mucosal adjuvant, with mucosal adjuvant LTB as fusion protein and OMP18 front end are connected in series in turn, the introduction of 5 amino acids in the segment linker PQDPP connection; to construct fusion gene in E.coli the expression vector pET-28a, restriction enzyme digestion and sequencing. The positive recombinant plasmid was transformed into host bacteria E.coliBL21 (DE3), the expression of.Tris-Tricine fusion protein electrophoresis and Western blot identification of recombinant fusion protein induced by IPTG, analysis of physicochemical properties of DNAssist software, the purification conditions explored through purification of AKTA-explore instrument, the fusion protein was purified by LTB-OMP18 (LO) and UreB414-OMP18-LTB (UOL). The purified fusion protein were combined with test and GM1 ganglioside.
3, 144 mice were randomly divided into 6 groups: PBS control group, single subunit intramocecular adjuvant fusion protein group (UreB414-LTB), double subunit intramocecular adjuvant fusion protein group (UreB414-OMP18-LTB) single subunit intramocecular adjuvant fusion protein group (LTB-OMP18), and the mixed group of external objects (rOMP18+UreB414-LTB) no intra molecular adjuvant single subunit protein group (OMP18). In 0,7,14,28 days of oral immunization, the dose of 150 g/ / time. In 10 days after immunization, 12 mice in each group were killed after oral immunization of ELISA sampling, detection of serum specific IgG, IgA, feces, intestinal flushing fluid sIgA levels. Each of the remaining 12 only 10~8 CFU Hp mice oral adapted strains, 30 days later, the mice stomach specimens by Hp culture and the specificity of PCR detection, to determine the infection rate of Hp, and calculate each group after oral immunization protection rate of virus attack.
Result:
1, by enzyme digestion and sequencing showed that the nucleotide sequence length of H.pylori OMP18 gene was correctly cloned into the pQE-30.OMP18 gene was 540bp, compared with GenBank sequence, the homology of 99% homologous amino acid sequences of 100%. recombinant IPTG induced by Tris-Tricine objective analysis, the molecular weight of the expressed protein about 20KD, the expression of the total bacterial protein 20%, expressed as the form of inclusion body, the 100 AKTA-explore protein purification system and purification, after purification the purity of target protein. In 85%, the purified protein as antigen to immune agent Fagafaga F Szo rabbit, preparation of Rabbit anti rOMP18 antibodies, by double immunodiffusion assay, rabbit antiserum the antibody titer was 1:32, Western Blot results also showed that the recombinant protein could be recognized by Rabbit anti H.pylori serum.
2, by enzyme digestion and sequencing results showed that the successful construction of the fusion gene of LTB-OMP18 and UreB414-OMP18-LTB, the fusion gene LO and UOL cloned into the pET28a vector, to obtain the fusion protein expression vector pLO and pUOL, linker protein evaluation software DnaStra has good flexibility. The two positive recombinants were transformed into E.coli (BL21 DE3), induced by IPTG 37 C culture, expression of the fusion protein by SDS-PAGE and Western blot analysis showed that the molecular weight of about 31KD and 43KD.UVP scan confirmed the expression of fusion protein accounted for about 25% of total protein and 20%. inclusion identification test confirmed the fusion protein expressed mainly in the form of inclusion bodies were determined by the method of affinity chromatography. The purified fusion protein purity > 85%. Two fusion protein after purification respectively binding reaction with GM1 ganglioside, experiments confirmed that the recombinant fusion protein components in LTB It has biological activity associated with GM1 ganglioside, indicating that the fusion protein maintains the natural activity of LTB and has the activity of the mucosal adjuvant.
3, detection of ELISA fusion protein rLO and rUOL in mice serum specific IgG, IgA, feces, intestinal flushing fluid sIgA levels compared with PBS group was significantly increased, significant difference (P < 0.001), compared with the subunit immune group, significant difference (P < 0.05), compared with the multi subunit protein physics the mixed group, the difference was not significant (P > 0.05). Immunity, immune protection rate was 67% (8/12): rLO group, rUOL group of 83% (10/12). Statistical analysis showed that the subunit fusion protein group immune protection rate compared with the PBS group, significant difference (P < 0.001), compared with rOMP18 group, significant difference (P < 0.05), compared with the rOMP18+UreB414-LTB group, the difference was not significant difference (P > 0.05).
Conclusion:
1, the H.pylori OMP18 gene has been cloned successfully, which is highly homologous with the H.pylori gene sequence published by GenBank. The purified recombinant protein rOMP18 has good immunogenicity and immunoreactivity, and it can be used as a candidate antigen for H.pylori genetic engineering vaccine.
2, the fusion protein rLO and rUOL were successfully constructed, and the purified fusion protein maintained independent immune response of each subunit component and adjuvant. The Linker between fusion genes had little effect on the three-dimensional conformation of fusion protein.
3, the level of specific antibody and immune protection in mice showed that the fusion protein rLO and rUOL had good safety and protective effects.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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