發(fā)布時間：2018-02-27 01:35

  本文關(guān)鍵詞： 幽門螺桿菌 外膜蛋白 OMP18 免疫保護 粘膜免疫　出處：《第三軍醫(yī)大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 幽門螺桿菌(H.pylori)的全球感染率超過了50%,它是慢性活動性胃炎和消化性潰瘍的主要病因,與腸型胃癌和胃黏膜相關(guān)淋巴樣組織(MALT)淋巴瘤的發(fā)生密切相關(guān)�，F(xiàn)有的根除H.pylori的手段主要是聯(lián)合應(yīng)用抗生素,但由于該菌感染的普遍性、耐藥菌株的不斷增加、較高的花費以及病人的低依從性等,限制了抗菌藥物的療效。因此,接種簡便、成本低廉并易于大面積推廣的H.pylori疫苗的研究十分迫切。 在疫苗研究中,篩選有效的免疫抗原是關(guān)鍵性的環(huán)節(jié)。目前大多數(shù)疫苗采用的是與Hp致病相關(guān)的毒力因子作為抗原成分,如尿素酶(urease)、熱休克蛋白(heat shockprotein,Hsp)、空泡毒素(vacuolating cytotoxin,VacA)、細胞毒素相關(guān)抗原(cytotoxinassociate antigen,CagA)、中性粒細胞激活蛋白(neutrophil-activating protein,NAP)等。革蘭氏陰性細菌外膜蛋白具有良好的抗原活性,這些外膜蛋白作為抗體和免疫細胞攻擊的主要靶標,可以介導(dǎo)對細菌最直接有效的殺滅作用,是決定免疫反應(yīng)是否對人體具有保護性的關(guān)鍵因素。OMP18和UerB都是Hp的外膜蛋白成分,其編碼基因具有高度保守性,也是Hp疫苗重要的免疫抗原。但一直令學(xué)者們遺憾的是每種抗原單獨免疫動物時,獲得的保護率均不是很高,而多種抗原聯(lián)合免疫才能達到較好的效果。 因此,本研究擬對上述二種保護性抗原基因進行克隆表達,并與分子內(nèi)佐劑LTB串聯(lián)得到多亞單位的融合蛋白,分別觀察在小鼠體內(nèi)誘導(dǎo)的不同免疫應(yīng)答和保護作用,為篩選有效的H.pylori疫苗抗原奠定基礎(chǔ)。 方法: 1、采用PCR方法從H.pylori臨床分離株9806基因組中擴增OMP18基因片段并構(gòu)建在原核表達載體pQE-30中,通過酶切、測序鑒定陽性重組子。應(yīng)用生物信息學(xué)軟件DNAssist和GenBank數(shù)據(jù)庫對已公布的H.pylori OMP18基因序列進行相似性分析。將含目的基因片段的重組載體轉(zhuǎn)化大腸桿菌M15中,經(jīng)IPTG誘導(dǎo)表達,用AKTA-explore純化儀純化表達的重組蛋白rOMP18。應(yīng)用Tris-Tricine方法對表達產(chǎn)物和純化產(chǎn)物進行分析,用純化的rOMP18免疫家兔,并采用Western blot和免疫擴散試驗鑒定重組蛋白的免疫性。 2、采用重疊延伸PCR的方法,分別以UreB414活性片段做為融合蛋白前端,與OMP18及粘膜佐劑LTB依次串聯(lián),和以粘膜佐劑LTB做為融合蛋白前端與OMP18依次串聯(lián),在片段間引入5個氨基酸的linker PQDPP進行連接;將融合基因構(gòu)建在原核表達載體pET-28a中,經(jīng)酶切及測序鑒定后,陽性重組子轉(zhuǎn)化宿主菌E.coliBL21(DE3),IPTG誘導(dǎo)重組工程菌表達。Tris-Tricine電泳和免疫印跡鑒定融合蛋白,DNAssist軟件分析融合蛋白理化特性,通過純化條件摸索,AKTA-explore純化儀純化融合蛋白LTB-OMP18(LO)和UreB414-OMP18-LTB(UOL)。純化后的融合蛋白分別與GM1神經(jīng)節(jié)苷脂進行結(jié)合試驗。 3、將144只小鼠隨機分為6組:PBS對照組、單亞單位分子內(nèi)佐劑融合蛋白組(UreB414-LTB)、雙亞單位分子內(nèi)佐劑融合蛋白組(UreB414-OMP18-LTB、)單一亞單位分子內(nèi)佐劑融合蛋白組(LTB-OMP18)、體外物理混合組(rOMP18+UreB414-LTB)無分子內(nèi)佐劑單一亞單位蛋白組(OMP18)。分別于0、7、14、28天口服免疫,劑量為150μg/只/次。術(shù)次免疫后10天,每組取12只小鼠剖殺取樣,ELISA檢測口服免疫后血清特異性IgG、IgA,糞便、腸道沖洗液sIgA水平。每組余下12只口服10~8 CFU Hp小鼠適應(yīng)株,30天后,取小鼠胃部標本通過Hp培養(yǎng)及特異性PCR檢測,確定Hp感染率,并計算口服免疫后各組攻毒保護率。 結(jié)果: 1、經(jīng)酶切鑒定和基因測序結(jié)果顯示,H.pylori OMP18基因被正確克隆到pQE-30中。OMP18基因的核苷酸序列長度為540bp,與GenBank中序列比較,同源性為99%,氨基酸序列的同源性為100%。重組工程菌IPTG誘導(dǎo),經(jīng)Tris-Tricine分析,表達的目的蛋白分子量約為20KD,其表達產(chǎn)物占菌體蛋白的20%,為包涵體形式表達,經(jīng)AKTA-explore 100蛋白純化系統(tǒng)純化,純化后得到純度＞85%的目的蛋白。以純化蛋白為抗原加弗氏佐劑免疫家兔,制備兔抗rOMP18特異抗體,經(jīng)免疫雙擴散法檢測,兔抗血清抗體效價為1:32,Western Blot結(jié)果也顯示重組蛋白能被兔抗H.pylori血清所識別。 2、經(jīng)酶切鑒定和基因測序結(jié)果顯示,成功構(gòu)建了融合基因LTB-OMP18和UreB414-OMP18-LTB,將融合基因LO和UOL克隆到pET28a載體上,得到融合蛋白表達載體pLO和pUOL,DnaStra軟件評價蛋白linker具有較好的柔韌性。將兩個陽性重組子轉(zhuǎn)化至E.coli BL21(DE3)后,37℃培養(yǎng)經(jīng)IPTG誘導(dǎo),表達的融合蛋白經(jīng)SDS-PAGE和免疫印跡分析顯示分子量約為31KD和43KD。UVP掃描證實融合蛋白表達約占總蛋白的25%和20%。包涵體鑒定試驗證實融合蛋白主要以包涵體形式表達,確定了采用親和層析的純化方法。純化后得到純度＞85%的融合蛋白。純化后的兩個融合蛋白分別能與GM1神經(jīng)節(jié)苷脂發(fā)生結(jié)合反應(yīng),實驗證實重組融合蛋白中LTB組分具有與GM1神經(jīng)節(jié)苷脂結(jié)合的生物活性,說明融合蛋白保持了LTB的天然活性,具有粘膜佐劑活性。 3、ELISA檢測融合蛋白rLO和rUOL免疫小鼠后血清特異性IgG、IgA,糞便、腸道沖洗液sIgA水平與PBS組相比,升高明顯,差異極顯著(p＜0.001),與亞單位免疫組相比,差異顯著(p＜0.05),與多亞單位蛋白物理混合組相比,差異不顯著(p＞0.05)。免疫攻毒后,免疫保護率為:rLO組67%(8/12),rUOL組83%(10/12)。統(tǒng)計分析顯示,多亞單位融合蛋白組免疫保護率與PBS組相比,差異極顯著(p＜0.001),與rOMP18組相比,差異顯著(p＜0.05),與rOMP18+UreB414-LTB組相比,差異不顯差(p＞0.05)。 結(jié)論: 1、成功克隆了H.pylori OMP18基因,與GenBank已公布的H.pylori菌株基因序列具有高度同源性。純化的重組蛋白rOMP18,具有良好的免疫原性和免疫反應(yīng)性,可以作為H.pylori基因工程疫苗候選抗原。 2、成功構(gòu)建、表達融合蛋白rLO和rUOL,純化后的融合蛋白保持各亞單位組分及佐劑的獨立免疫反應(yīng)性,融合基因間的Linker對融合蛋白的立體構(gòu)象影響較小。 3、小鼠體內(nèi)特異性抗體水平及免疫保護效果都證明融合蛋白rLO和rUOL具有良好的安全性和免疫保護效果。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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