本文關(guān)鍵詞：屋塵螨抗原Der p2重組恥垢分枝桿菌口服疫苗的制備及其實(shí)驗(yàn)研究　出處：《第四軍醫(yī)大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 屋塵螨抗原 Der p2 重組 恥垢分枝桿菌 口服疫苗 PEB1

【摘要】： 支氣管哮喘(Asthma)是嚴(yán)重威脅人類健康的一種常見慢性疾病,其發(fā)病率和死亡率在世界各地呈逐年上升的趨勢,糖皮質(zhì)激素雖能有效地控制其癥狀,但因其長期使用會出現(xiàn)不良反應(yīng),以及不能糾正機(jī)體已有的免疫紊亂和阻斷疾病進(jìn)程,因此臨床上迫切需要尋找有效的途徑來預(yù)防此類變態(tài)反應(yīng)性疾病的發(fā)生與發(fā)展。研究表明,變態(tài)反應(yīng)性疾病患者體內(nèi)呈現(xiàn)過度的Th2型免疫應(yīng)答,而結(jié)核桿菌等分枝桿菌感染可以誘導(dǎo)機(jī)體產(chǎn)生強(qiáng)烈的Thl型免疫應(yīng)答。為改變機(jī)體對某些過敏原Th2優(yōu)勢應(yīng)答的狀態(tài),我科曾成功將屋塵螨抗原Der p2基因轉(zhuǎn)入卡介苗(BCG)中表達(dá),制備出抗原重組BCG(rBCG)。經(jīng)靜脈和腹腔注射接種后,在小鼠體內(nèi)誘導(dǎo)了Der p2特異性的Th1優(yōu)勢應(yīng)答。這意味著象哮喘這類對某些特定抗原過敏的疾病,可以利用抗原rBCG作為疫苗而得到治療。 但是短期內(nèi)重復(fù)注射接種rBCG會引起局部嚴(yán)重的遲發(fā)性變態(tài)反應(yīng)而影響療效。為此,進(jìn)一步給小鼠口服Der p2-rBCG,結(jié)果同樣誘導(dǎo)了抗原特異性的Th1應(yīng)答。相關(guān)研究表明,口服分枝桿菌疫苗不僅能夠刺激理想的免疫應(yīng)答,而且經(jīng)濟(jì)方便。然而,分枝桿菌不是腸道定植菌,其與腸道粘膜的親和力過低,大量口服還會引起腸道正常菌群失調(diào)和口咽部感染,從而影響免疫效果。研究發(fā)現(xiàn),空腸彎曲菌(C.jejuni)外膜蛋白PEB1是C.jejuni的主要粘附分子,在C.jejuni與腸道粘膜的粘附中發(fā)揮重要作用,這為制備抗原特異性的腸道高親和力重組分枝桿菌口服疫苗創(chuàng)造了條件。 目的 1.利用基因工程手段,構(gòu)建能以胞壁形式表達(dá)屋塵螨Der p2和C.jejuni PEB1抗原的重組恥垢分枝桿菌(rM.S)口服疫苗Der p2-PEB1-rM.S。 2.將Der p2-PEB1-rM.S與Der p2-rM.S一起口服免疫小鼠,比較兩者的生物學(xué)行為及免疫學(xué)特性,為臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。 實(shí)驗(yàn)方法和結(jié)果 1. PEB1蛋白的表達(dá)及純化 以C.jejuni Penner O:19標(biāo)準(zhǔn)株基因組為模板,用PCR法擴(kuò)增PEB1基因,并與pUC19克隆載體連接,經(jīng)測序證實(shí)與Genbank公布的C.jejuni PEB1基因序列完全一致。將PEB1基因克隆入pPRO EX HTb原核表達(dá)載體,酶切鑒定陽性重組質(zhì)粒pPRO-PEB1,并用IPTG進(jìn)行誘導(dǎo)表達(dá)。SDS-PAGE分析表明成功地表達(dá)了與預(yù)期分子量一致的PEB1融合蛋白;Western-blot證實(shí)該蛋白可與抗His-mAb發(fā)生特異性反應(yīng)。可溶性分析顯示該蛋白以可溶形式存在于上清中,用Ni-NTA親和色譜法純化獲得了目的蛋白,純度達(dá)90%以上。 2.小鼠抗PEB1蛋白多克隆抗體的制備 用皮下包埋的方法,以預(yù)先轉(zhuǎn)移到硝酸纖維素膜上的純化PEB1蛋白免疫BALB/c小鼠,共免疫三次,每次間隔2周,最后一次免疫結(jié)束2周后收集小鼠血清,同時(shí)設(shè)生理鹽水對照。間接ELISA方法測定免疫小鼠血清中PEB1蛋白特異性抗體滴度為1:204800。 3.胞壁表達(dá)Der p2-PEB1融合蛋白重組恥垢分枝桿菌的構(gòu)建和鑒定 用PCR法分別擴(kuò)增PEB1和Der p2基因,經(jīng)測序正確后按照相應(yīng)的酶切位點(diǎn)將兩者克隆入pUC19,得到Der p2-PEB1融合基因。將Der p2-PEB1融合基因亞克隆入大腸埃希菌-分枝桿菌穿梭胞壁表達(dá)載體pCW,構(gòu)建可胞壁表達(dá)Der p2-PEB1融合蛋白的重組質(zhì)粒pCW-Der p2-PEB1,并經(jīng)酶切鑒定證實(shí)。將重組質(zhì)粒電穿導(dǎo)入M.S感受態(tài)細(xì)胞,經(jīng)潮霉素抗性篩選rM.S陽性克隆。陽性rM.S經(jīng)PCR特異性擴(kuò)增目的基因片段,證實(shí)其中含有目的基因。用抗Der p2單克隆抗體和制備的小鼠PEB1抗血清對rM.S進(jìn)行間接免疫熒光鑒定,證實(shí)Der p2-PEB1融合蛋白可以在M.S表面表達(dá)。 4.消化道環(huán)境對重組M.S疫苗生物學(xué)行為的影響 分別以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,連續(xù)5天。于首次免疫后第1、6、8、10、14、28、56d收集各組小鼠糞便,處理后涂布于7H10瓊脂平板,培養(yǎng)后計(jì)數(shù)平板上M.S的CFU。分別于末次免疫后第2、14、28、56d處死小鼠,無菌分離腸道相關(guān)淋巴組織(GALT)、脾臟和肺臟,勻漿后涂布7H10瓊脂平板,計(jì)數(shù)平板上M.S的CFU。結(jié)果顯示,各組小鼠于口服免疫后次日即可在其糞便中排出M.S,第6天最多,隨后逐漸減少,至免疫后2周完全消失,各組之間并無差異。小鼠口服免疫結(jié)束后第2、14、28天均能在GALT勻漿中檢出M.S,其中pCW-Der p2-PEB1-rM.S組各時(shí)間點(diǎn)菌落數(shù)均較pCW-Der p2-rM.S組明顯增加(P0.05)。除第56天外,各組小鼠在其余各時(shí)間點(diǎn)均能在肺中檢出少量M.S。而所有小鼠在各個(gè)時(shí)間點(diǎn)均未從脾臟中檢出M.S。上述結(jié)果表明,PCW-Der p2-PEB1-rM.S更易于被腸道粘膜攝取并進(jìn)入GALT。 5.不同親和力rM.S口服接種刺激BALB/c小鼠免疫應(yīng)答的差異 分別予以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,連續(xù)5天。于末次免疫后14、28、56天將小鼠摘眼球取血,收集血清,并制備脾淋巴細(xì)胞培養(yǎng)上清,用夾心ELISA法分別測定其中IFN-γ、IL-2和IL-4水平。結(jié)果表明,兩種疫苗免疫后小鼠血清和脾淋巴細(xì)胞培養(yǎng)上清中Th1型細(xì)胞因子IFN-γ、IL-2水平均明顯升高,Th2型細(xì)胞因子IL-4水平則明顯下降;在小鼠脾淋巴細(xì)胞培養(yǎng)上清中加入Der p2刺激可使IFN-γ、IL-2含量升高、IL-4含量降低較M.S組更為明顯;兩種rM.S之間的比較也發(fā)現(xiàn),pCW-Der p2-PEB1-rM.S組較pCW-Der p2-rM.S組IFN-γ、IL-2水平有明顯升高,而IL-4水平無明顯差異。這說明:rM.S免疫后在小鼠體內(nèi)引發(fā)的免疫反應(yīng)以誘導(dǎo)Th1優(yōu)勢為主,且這種應(yīng)答是Der p2特異性的;PEB1導(dǎo)入rM.S后可以提高rM.S口服的免疫效率。 結(jié)論: 1.兩種不同親和力的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過消化道進(jìn)入GALT;融合了PEB1蛋白后,消化道粘膜對rM.S的攝取量有所增加。 2.它們免疫后在小鼠體內(nèi)引發(fā)的免疫應(yīng)答具有Th1優(yōu)勢的特征,且這種Th1優(yōu)勢是Der p2抗原特異性的;PEB1導(dǎo)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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392

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