【作者】 简文；

【导师】 戚好文； 史皆然；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 支气管哮喘(Asthma)是严重威胁人类健康的一种常见慢性疾病,其发病率和死亡率在世界各地呈逐年上升的趋势,糖皮质激素虽能有效地控制其症状,但因其长期使用会出现不良反应,以及不能纠正机体已有的免疫紊乱和阻断疾病进程,因此临床上迫切需要寻找有效的途径来预防此类变态反应性疾病的发生与发展。研究表明,变态反应性疾病患者体内呈现过度的Th2型免疫应答,而结核杆菌等分枝杆菌感染可以诱导机体产生强烈的Thl型免疫应答。为改变机体对某些过敏原Th2优势应答的状态,我科曾成功将屋尘螨抗原Der p2基因转入卡介苗(BCG)中表达,制备出抗原重组BCG(rBCG)。经静脉和腹腔注射接种后,在小鼠体内诱导了Der p2特异性的Th1优势应答。这意味着象哮喘这类对某些特定抗原过敏的疾病,可以利用抗原rBCG作为疫苗而得到治疗。但是短期内重复注射接种rBCG会引起局部严重的迟发性变态反应而影响疗效。为此,进一步给小鼠口服Der p2-rBCG,结果同样诱导了抗原特异性的Th1应答。相关研究表明,口服分枝杆菌疫苗不仅能够刺激理想的免疫应答,而且经济方便。然而,分枝杆菌不是肠道定植菌,其与肠道粘膜的亲和力过低,大量口服还会引起肠道正常菌群失调和口咽部感染,从而影响免疫效果。研究发现,空肠弯曲菌(C.jejuni)外膜蛋白PEB1是C.jejuni的主要粘附分子,在C.jejuni与肠道粘膜的粘附中发挥重要作用,这为制备抗原特异性的肠道高亲和力重组分枝杆菌口服疫苗创造了条件。目的1.利用基因工程手段,构建能以胞壁形式表达屋尘螨Der p2和C.jejuni PEB1抗原的重组耻垢分枝杆菌(rM.S)口服疫苗Der p2-PEB1-rM.S。2.将Der p2-PEB1-rM.S与Der p2-rM.S一起口服免疫小鼠,比较两者的生物学行为及免疫学特性,为临床应用提供实验依据。实验方法和结果1. PEB1蛋白的表达及纯化以C.jejuni Penner O:19标准株基因组为模板,用PCR法扩增PEB1基因,并与pUC19克隆载体连接,经测序证实与Genbank公布的C.jejuni PEB1基因序列完全一致。将PEB1基因克隆入pPRO EX HTb原核表达载体,酶切鉴定阳性重组质粒pPRO-PEB1,并用IPTG进行诱导表达。SDS-PAGE分析表明成功地表达了与预期分子量一致的PEB1融合蛋白;Western-blot证实该蛋白可与抗His-mAb发生特异性反应。可溶性分析显示该蛋白以可溶形式存在于上清中,用Ni-NTA亲和色谱法纯化获得了目的蛋白,纯度达90%以上。2.小鼠抗PEB1蛋白多克隆抗体的制备用皮下包埋的方法,以预先转移到硝酸纤维素膜上的纯化PEB1蛋白免疫BALB/c小鼠,共免疫三次,每次间隔2周,最后一次免疫结束2周后收集小鼠血清,同时设生理盐水对照。间接ELISA方法测定免疫小鼠血清中PEB1蛋白特异性抗体滴度为1:204800。3.胞壁表达Der p2-PEB1融合蛋白重组耻垢分枝杆菌的构建和鉴定用PCR法分别扩增PEB1和Der p2基因,经测序正确后按照相应的酶切位点将两者克隆入pUC19,得到Der p2-PEB1融合基因。将Der p2-PEB1融合基因亚克隆入大肠埃希菌-分枝杆菌穿梭胞壁表达载体pCW,构建可胞壁表达Der p2-PEB1融合蛋白的重组质粒pCW-Der p2-PEB1,并经酶切鉴定证实。将重组质粒电穿导入M.S感受态细胞,经潮霉素抗性筛选rM.S阳性克隆。阳性rM.S经PCR特异性扩增目的基因片段,证实其中含有目的基因。用抗Der p2单克隆抗体和制备的小鼠PEB1抗血清对rM.S进行间接免疫荧光鉴定,证实Der p2-PEB1融合蛋白可以在M.S表面表达。4.消化道环境对重组M.S疫苗生物学行为的影响分别以100μl pCW-Der p2-PEB1-rM.S、pCW-Der p2-rM.S和M.S(1×1013 CFU·L-1)灌胃免疫BALB/c小鼠,连续5天。于首次免疫后第1、6、8、10、14、28、56d收集各组小鼠粪便,处理后涂布于7H10琼脂平板,培养后计数平板上M.S的CFU。分别于末次免疫后第2、14、28、56d处死小鼠,无菌分离肠道相关淋巴组织(GALT)、脾脏和肺脏,匀浆后涂布7H10琼脂平板,计数平板上M.S的CFU。结果显示,各组小鼠于口服免疫后次日即可在其粪便中排出M.S,第6天最多,随后逐渐减少,至免疫后2周完全消失,各组之间并无差异。小鼠口服免疫结束后第2、14、28天均能在GALT匀浆中检出M.S,其中pCW-Der p2-PEB1-rM.S组各时间点菌落数均较pCW-Der p2-rM.S组明显增加(P<0.05)。除第56天外,各组小鼠在其余各时间点均能在肺中检出少量M.S。而所有小鼠在各个时间点均未从脾脏中检出M.S。上述结果表明,PCW-Der p2-PEB1-rM.S更易于被肠道粘膜摄取并进入GALT。5.不同亲和力rM.S口服接种刺激BALB/c小鼠免疫应答的差异分别予以100μl pCW-Der p2-PEB1-rM.S、pCW-Der p2-rM.S、M.S(1×1013 CFU·L-1)和甘油灌胃免疫BALB/c小鼠,连续5天。于末次免疫后14、28、56天将小鼠摘眼球取血,收集血清,并制备脾淋巴细胞培养上清,用夹心ELISA法分别测定其中IFN-γ、IL-2和IL-4水平。结果表明,两种疫苗免疫后小鼠血清和脾淋巴细胞培养上清中Th1型细胞因子IFN-γ、IL-2水平均明显升高,Th2型细胞因子IL-4水平则明显下降;在小鼠脾淋巴细胞培养上清中加入Der p2刺激可使IFN-γ、IL-2含量升高、IL-4含量降低较M.S组更为明显;两种rM.S之间的比较也发现,pCW-Der p2-PEB1-rM.S组较pCW-Der p2-rM.S组IFN-γ、IL-2水平有明显升高,而IL-4水平无明显差异。这说明:rM.S免疫后在小鼠体内引发的免疫反应以诱导Th1优势为主,且这种应答是Der p2特异性的;PEB1导入rM.S后可以提高rM.S口服的免疫效率。结论:1.两种不同亲和力的rM.S疫苗pCW-Der p2-PEB1-rM.S和pCW-Der p2-rM.S口服免疫小鼠后均可通过消化道进入GALT;融合了PEB1蛋白后,消化道粘膜对rM.S的摄取量有所增加。2.它们免疫后在小鼠体内引发的免疫应答具有Th1优势的特征,且这种Th1优势是Der p2抗原特异性的;PEB1导入rM.S后可以提高rM.S口服的免疫效率。更多还原

【Abstract】 Asthma is a serious public health problem, affecting people of all ages. The morbidity and mortality of asthma is increasing throughout the world in recent years. Although glucocorticoid can effectively reduce its symptoms, the strong side effects limited its use in medical practice. Furthermore, it can not adjust immunological disorders and impede the progressing of asthma. So, it is urgent in clinical practice to find effective ways to prevent the occurrence and development of this allergic disease. Studies showed that there was excessive Th2 immune response in patients with allergic disease, while mycobacterium such as mycobacterium tuberculosis might induce predominant Th1 immune response. In order to change the predominant Th2 immune response induced by some anaphylactogen, we constructed recombinant BCG by transferring Der p2 into BCG in our lab. The recombinant Der p2-BCG could stimulate Th1 predominant immune response in mice, when injected intraperitoneally or subcutaneously. The results indicated that antigen recombinant BCG could be used to treat asthma which was hypersensitive to some specific antigen.Given that the repeated injection of rBCG vaccine could induce serious local delayed-type hypersensitivity, which might influence the therapeutic efficacy, oral vaccine was administered to mice. Results showed that it could also induce antigen specific Th1 predominant immune response. Related studies indicated that oral vaccination of mycobacterium was not only effective in stimulating ideal immune response, but also economic and convenient. Mycobacterium usually doesn’t inhabit in the intestinal tract because of its low affinity to intestinal epithelia. Furthermore, a large dosis of mycobacteria could cause dysbacteria and oropharynx adenitis. So its immune efficacy is limited. Protein PEB1 was found to be one of the major adhesion molecules which played an important role in adhesion between Campylobacter jejuni (C.jejuni) and intestinal mucosa. These findings provide us with a convincing theory of constructing antigen specific oral vaccine of recombinant mycobacterium.AIM1. To construct the oral recombinant mycobacterium smegmatis (M.S) vaccine expressing the fusion protein of Der p2 and PEB1 on its cell wall.2. To Compare Der p2-PEB1-rM.S vaccine with Der p2-rM.S vaccine by evaluating their differential biological behaviors and immunological properties.METHODS AND RESULTS1. Expression and purification of protein PEB1PEB1 gene was amplified by polymerase chain reaction (PCR) from genomic DNA of M.S, and cloned into vector pUC19. The DNA sequence of PEB1 was identical with that published in GenBank. After confirmed by sequencing, the target gene was subcloned into prokaryotic expression vector pProEXHTb and identified by restrictive enzyme digestion. The recombinant plasmid was then transformed into E.coli DH5αstrain and induced by IPTG. The analysis of SDS-PAGE showed that there was a specific protein expression at 28kD molecular marker just as we anticipated, and the protein was further identified by Western-blot using anti-His mAb. The fusion protein was purified by Ni-NTA purification system under native condition and its purity was about ninety percent.2. Preparation of PEB1 polyclonal antibody in miceBALB/c mice were immunized subcutaneously three times at 2-week intervals, by embedded in fold groin with recombinant protein loaded to nitrocellulose filter. The control group were given 0.9% saline. Antibody titers of PEB1 protein group increased the maximum at 1: 204800 after 6 weeks of the first vaccination detected by ELISA.3. Construction and expression of the recombinant M.S expressing Der p2-PEB1 fusion proteinThe genes of Der p2 and PEB1 were first amplified by PCR respectively, then ligated into pUC19 vector. At last, the Der p2-PEB1 fused gene was subcloned into the E.coli-mycobacterium shuttle plasmid pCW. The recombinant plasmid was identified by restrictive enzyme digestion and the positive plasmid was named pCW-Der p2-PEB1. The plasmid was transformed into M.S competent cells and selected by hygromycine. The positive rM.S was identified by PCR and the result showed that there was specific amplification of the target gene. Indirect immunofluorescence was used to detect the fusion protein expressed in bacterium body of rM.S, and specifc green immunofluorescence was observed, indicating that rM.S could express the goal protein on its surface.4. Influence of intestinal tract environment on biological behavior of rM.S vaccineBALB/c mice were immunized continuously with 100μl pCW-Der p2-PEB1-rM.S, pCW-Der p2-rM.S and M.S(1×1013 CFU·L-1) via oral administration for 5 days respectively. Feces of every group were collected at 1, 6, 8, 10, 14, 28, 56 days after the first vaccination. The handled feces were plated and incubated on 7H10 agar plates, and the bacteria numbers were counted. Mice were killed at 2, 14, 28, 56 days after vaccination. Organs such as GALT, lungs and spleens were isolated aseptically, then homogenized and plated on 7H10 agar plates, at last the bacteria numbers were counted. The results showed that M.S were found in feces of every group mice in the second day after oral immunization. The numbers of M.S achieved the maximum at the sixth day, and then gradually decreased in the following days, at last disappeared in the second week after immunization. There was no significant difference among the three groups. M.S could be identified from homogenates of GALT at 2, 14 and 28 days after immunization, and the numbers of pCW-Der p2-PEB1-rM.S group were higher than that of pCW-Der p2-rM.S group at all time points ( P<0.05 ) . Few bacteria were identified from homogenates of lungs in all groups at all time points except 56 days after immunization. No live mycobacterium was recovered from the spleens in all groups. These results indicated that pCW-Der p2-PEB1-rM.S could be easily intaken by intestinal epithelia and could reach into GALT.5. The immune responses induced by oral rM.S with different affinity in miceBALB/c mice were immunized continuously with 100μl pCW-Der p2-PEB1-rM.S, pCW-Der p2-rM.S, M.S(1×1013CFU·L-1) and glycerol via oral administration for 5 days respectively. Mice blood sera were collected and culture supernatant of spleen lymphocytes were prepared at 14, 28, and 56 days after the last immunization, and the levels of IFN-γ, IL-2 and IL-4 were detected respectively by ELISA. Results showed that the levels of IFN-γand IL-2 from sera and the supernatant of cultured spleen lymphocytes induced by these two rM.S vaccines were significantly high, while the levels of IL-4 were significantly low. Similarly, the Der p2 stimulation could induce higher levels of IFN-γ, IL-2 and lower levels of IL-4 than those of M.S group in culture supernatant of spleen lymphocytes. Comparing the two rM.S groups, we also found that the concentrations of IFN-γand IL-2 in pCW-Der p2-PEB1-rM.S group were significantly higher than those in pCW-Der p2-rM.S group, but the concentrations of IL-4 had no difference between the two groups. We can conclude that -11- the vaccination of rM.S could induce the Der p2 specific Th1 predominant immune response, and the transformation of PEB1 to rM.S could increase the efficacy of oral vaccination of rM.S.CONCLUSION1. Both of the two rM.S vaccines with different affinity could be intaken by intestinal epithelia and reach into GALT in mice after oral vaccinition. Intestinal epithelial cells could ingested more rM.S, when it was fused with PEB1 protein.2. Both of the rM.S vaccines could induce Der p2 specific Th1 predominant immune response in mice after oral vaccinition, and the transformation of PEB1 to rM.S could elevate the efficacy of oral rM.S vaccines.更多还原