屋尘螨抗原Der p2重组耻垢分枝杆菌口服疫苗的制备及其实验研究

发布时间：2018-01-01 16:11

本文关键词：屋尘螨抗原Der p2重组耻垢分枝杆菌口服疫苗的制备及其实验研究　出处：《第四军医大学》2008年博士论文　论文类型：学位论文

【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(P0.05)。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。
[Abstract]:Bronchial asthma (Asthma) is a serious threat to human health is a common chronic disease, the incidence and mortality rate showed an increasing trend in the world, although glucocorticoid can effectively control the symptoms, but because of the long-term use will lead to adverse reactions, and can not correct the body of existing immune disorders and blocking the disease process therefore, the clinical occurrence and development of urgent need to find effective ways to prevent such allergic diseases. The study shows that in patients with allergic disease showed Th2 type immune response over the Mycobacterium tuberculosis mycobacterium infection and so can induce a strong immune response. Thl to change the state of the body of certain allergies the original Th2 response, our department has successfully house dust mite antigen Der P2 gene was transformed into BCG (BCG) expression, preparation of recombinant BCG antigen (rBCG) by vein and abdominal. After vaccination, the Der P2 specific Th1 predominance response was induced in mice. This means that asthma, which is allergic to some specific antigens, can be treated with antigen rBCG as vaccine.
But in the short term repeated injection of rBCG vaccination may cause local severe delayed allergic reaction and the curative effect. Therefore, further to mice orally Der p2-rBCG, also induced the antigen-specific Th1 response. The research shows that the immune response of oral bacillus vaccine can not only stimulate the ideal, but also economic and convenient. However, Mycobacterium not intestinal colonization, and the intestinal mucosal affinity is too low, will cause a large number of oral and oropharyngeal dysbacteriosis of normal intestinal bacteria infection, thus affecting the immune effect. The study found that Campylobacter jejuni (C.jejuni) outer membrane protein PEB1 is the main adhesion molecule C.jejuni, plays an important role in C.jejuni and intestinal mucosal adhesion and this creates the conditions for preparation of high affinity intestinal Mycobacterium vaccine recombinant antigen specificity.
objective
1., we use genetic engineering to construct recombinant Der vaccine of Mycobacterium tuberculosis (rM.S), which can express house dust mite Der P2 and C.jejuni PEB1 antigen in cell wall form.
2. the mice were immunized with Der p2-PEB1-rM.S and Der p2-rM.S, and their biological behavior and immunological characteristics were compared to provide experimental basis for clinical application.
Experimental methods and results
Expression and purification of 1. PEB1 protein
The Penner O:19 strain genomic C.jejuni as template, PEB1 gene was amplified by PCR, and inserted into pUC19 vector, confirmed by sequencing C.jejuni PEB1 gene sequence is completely consistent with the published Genbank. PEB1 gene was cloned into pPRO EX prokaryotic expression vector HTb, enzyme digestion of recombinant plasmid pPRO-PEB1 Jian Dingyang, and the induced expression of.SDS-PAGE the analysis shows that the successful expression with the predicted molecular weight of PEB1 fusion protein with IPTG; Western-blot confirmed that the protein can specifically react with anti His-mAb. The analysis shows that the soluble protein in soluble form in the supernatant to obtain a protein purified by Ni-NTA affinity chromatography, the purity is more than 90%.
Preparation of anti PEB1 protein polyclonal antibody in 2. mice
The embedding method is used to transfer to the subcutaneous, pre purified PEB1 protein to immunize BALB/c mice nitrocellulose membrane, were immunized three times, each time interval of 2 weeks, last 2 weeks after the end of the collection of immune mouse serum, normal saline control group. The indirect ELISA method for determination of PEB1 protein specific immune serum antibody titer of small in the rat 1:204800.
Construction and identification of recombinant Mycobacterium tumefaciens with 3. cell walls expressing Der p2-PEB1 fusion protein
PEB1 and Der P2 gene were amplified by PCR method, after sequence according to the corresponding restriction sites both cloned into pUC19, Der p2-PEB1 Der p2-PEB1 fusion gene. The fusion gene was subcloned into Escherichia coli Mycobacterium shuttle expression vector pCW to construct cell wall, cell wall can express Der p2-PEB1 fusion protein the recombinant plasmid pCW-Der p2-PEB1, and confirmed by restriction endonuclease analysis. The recombinant plasmid electroporation into M.S competent cells with hygromycin resistance were rM.S positive clones. The positive rM.S by PCR amplification gene fragments containing target genes confirmed. PEB1 mice antiserum with anti Der P2 monoclonal antibody and preparation indirect immunofluorescence identification of rM.S, confirmed that the Der p2-PEB1 fusion protein can be expressed on the surface of M.S.
The effect of 4. digestive tract environment on the biological behavior of recombinant M.S vaccine
In 100 L pCW-Der p2-PEB1-rM.S, pCW-Der p2-rM.S and M.S (1 x 1013 CFU / L-1) orally immunized BALB/c mice for 5 days. In the first immunization after 1,6,8,10,14,28,56d were collected after treatment of mice feces, coated on 7H10 agar plate culture counting plate M.S, CFU. respectively after the last immunization No. 2,14,28,56d mice were sacrificed, sterile separation of gut associated lymphoid tissue (GALT), spleen and lung homogenate after coating, 7H10 agar plate count, M.S CFU. results showed that the mice after oral immunization of the M.S can be discharged in the feces, up to sixth days, and then decreased gradually until 2 weeks after immunization completely disappeared that there is no difference between the groups. Oral immunization in mice after 2,14,28 days were detected in M.S GALT in the homogenate, where pCW-Der p2-PEB1-rM.S group at each time point counts were lower than pCW-Der group p2-rM.S significantly increased (P0.05) in addition. Fifty-sixth days later, each group of mice could detect a small amount of M.S. in the lung at other time points, but all mice did not detect M.S. from spleen at any time point. The above results showed that PCW-Der p2-PEB1-rM.S was more easily absorbed by intestinal mucosa and entered GALT..
Difference of immune response in BALB/c mice stimulated by 5. different affinity rM.S oral inoculation
Respectively 100 L pCW-Der p2-PEB1-rM.S, pCW-Der p2-rM.S, M.S (1 x 1013 CFU / L-1) and glycerol orally immunized BALB/c mice for 5 days. On the day after the last immunization of 14,28,56 mice eyeball blood serum was collected, and the preparation of spleen lymphocyte supernatants were measured by sandwich IFN- gamma ELISA, IL-2 and IL-4 levels. The results showed that two kinds of vaccine and serum of mice spleen lymphocytes cultured in the supernatant of Th1 type cytokines IFN-, IL-2 levels were significantly increased, the level of Th2 type cytokines IL-4 decreased; in the accession to the Der in the supernatant of P2 stimulated IFN- gamma splenic lymph cells of mice cultured. The IL-2 content increased, IL-4 content decreased in M.S group was more obvious; the comparison between the two rM.S also found that pCW-Der p2-PEB1-rM.S group than in pCW-Der group p2-rM.S IFN- gamma, IL-2 levels increased significantly, while the level of IL-4 showed no significant difference. This shows that: rM.S after immunization The immune response initiated in mice is mainly induced by Th1, and this response is Der P2 specific. PEB1 can enhance the oral immune efficiency of rM.S after the introduction of rM.S.
Conclusion:
1., two kinds of rM.S vaccines with different affinity, pCW-Der p2-PEB1-rM.S and pCW-Der p2-rM.S orally immunized mice, could enter GALT through the digestive tract. After the fusion of PEB1 protein, the uptake of rM.S in digestive tract mucosa increased.
2., the immune response induced by immunization in mice has the advantage of Th1 dominance, and the Th1 advantage is Der P2 antigen specificity. PEB1 can improve the oral immune efficiency of rM.S after the introduction of rM.S.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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