本文選題：幽門螺桿菌 + 乳酸乳球菌　； 參考：《鄭州大學(xué)》2013年碩士論文

【摘要】：目的 對本課題組前期構(gòu)建好的H.pylori重組疫苗菌株進(jìn)行驗(yàn)證、誘導(dǎo)、表達(dá)。通過口服灌胃免疫接種BALB/c小鼠,連續(xù)免疫一個(gè)月后,用H.pylori國際標(biāo)準(zhǔn)株11637進(jìn)行攻擊,檢測小鼠胃內(nèi)H.pylori定植量,評價(jià)不同疫苗所產(chǎn)生的免疫應(yīng)答和免疫保護(hù)效果。 方法 1.將過夜培養(yǎng)好的乳酸菌NZ3900/pNZ8110-ureB、NZ3900/pNZ8110-lpp20、 NZ3900/pNZ8110-omp22-hpaA、NZ3900/pNZ8149-ureB、NZ3900/pNZ8110-LysM-hpaA、NZ3900/pNZ8149-SPusp45-ureB nisin誘導(dǎo)表達(dá),離心提取相關(guān)蛋白,經(jīng)SDS-PAGE電泳鑒定重組菌免疫蛋白表達(dá)水平,Western-blot鑒定重組蛋白免疫反應(yīng)性。 2.將過夜培養(yǎng)好的基因工程重組菌TB1(pMAL-c2X-ureB)、TB1(pMAL-c2X-hpaA)、TB1(pMAL-c2X-lpp20)、TB1(pMAL-c2X-omp22)IPTG誘導(dǎo)表達(dá)超聲破碎提取上清蛋白,經(jīng)SDS-PAGE驗(yàn)證后,應(yīng)用直鏈淀粉親和層析柱進(jìn)行純化,使用BCA蛋白定量試劑盒測定純化蛋白的濃度。 3.將BALB/c小鼠隨機(jī)分為9組,口服免疫后,收集一半小鼠的血清和胃液,酶聯(lián)免疫吸附試驗(yàn)(ELISA)法檢測特異性抗體含量水平。剩下小鼠用H.pylori進(jìn)行灌胃攻擊,2周后處死小鼠,取小鼠胃組織做尿素酶半定量實(shí)驗(yàn)。 4.統(tǒng)計(jì)學(xué)分析 應(yīng)用SPSS17.0進(jìn)行統(tǒng)計(jì)分析。各組資料進(jìn)行正態(tài)性檢驗(yàn)后,采用單因素方差分析對各組之間保護(hù)率進(jìn)行評價(jià)。 結(jié)果 1.SDS-PAGE膠照片顯示Nisin誘導(dǎo)重組蛋白Lpp20、UreB、Omp22-HpaA、 LysM-HpaA分別在19kd、66kd、53kd、29kd處顯示雜交條帶,Western-blot顯示各重組蛋白均有良好的免疫原性和免疫反應(yīng)性。 2.各基因工程重組菌誘導(dǎo)表達(dá)經(jīng)直鏈淀粉親和層析柱純化后,SDS-PAGE顯示在相應(yīng)條帶均有雜交條帶。 3.BALB/c小鼠口服灌胃后,NZ3900/pNZ8110-LysM-hpaA組血清IgG含量最高,其余各重組菌株組均高于陰性對照組(P0.05)。H.pylori灌胃攻擊后,快速尿素酶檢法顯示：各重組乳酸菌組OD450值均低于對照組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 1.獲得了純度較高H.pylori重組抗原UreB、Lpp20、HpaA、Omp22。 2.經(jīng)口服灌胃均具有免疫反應(yīng)性。 3.NZ3900/pNZ8110-LysM-hpaA所引起免疫保護(hù)效果最佳
[Abstract]:PurposeThe recombinant H.pylori vaccine strains constructed by our group were verified, induced and expressed.BALB/c mice were immunized by oral administration for one month, then attacked with H.pylori international standard strain 11637 to detect the quantity of H.pylori colonization in the stomach of mice, and to evaluate the immune response and protective effect of different vaccines.Method1.NZ3900 / pNZ8110-ureBNZ3900 / pNZ8110-lpp20, NZ3900 / pNZ8110-omp22-hpaAn NZ3900 / pNZ8149-ureBNZ3900p / pNZ8110-LysM-hpaAn NZ3900pNZ8149-SPusp45-ureB nisin was induced, and the protein expression level was identified by SDS-PAGE electrophoresis.2.The recombinant gene engineering strain TB1, pMAL-c2X-ureBHPAA, TB1, pMAL-c2X-hPAA, TB1, pMAL-c2X-lpp20, TB1, pMAL-c2X-omp22, was induced to extract the protein by ultrasonic fragmentation. The protein was purified by SDS-PAGE and purified by amylose affinity chromatography. The concentration of purified protein was determined by BCA protein quantitative assay kit.3.BALB/c mice were randomly divided into 9 groups. After oral immunization, the serum and gastric juice of half of the mice were collected and the specific antibody levels were detected by enzyme linked immunosorbent assay (Elisa).The rest of the mice were killed by H.pylori for 2 weeks, and the gastric tissues of the mice were taken for the semi-quantitative test of urease.4.Statistical analysisSPSS17.0 was used for statistical analysis.After normality test, the protection rate of each group was evaluated by single factor analysis of variance (ANOVA).Result1.SDS-PAGE gel photos showed that Nisin induced recombinant protein Lpp20UreBnOmp22-HpaA, and LysM-HpaA at 19kd ~ 66kd ~ 53kdU ~ (29) kd showed that all recombinant proteins showed good immunogenicity and immunoreactivity by Western-blot.2.After purification by amylose affinity chromatography, SDS-PAGE showed that there were hybridization bands in the corresponding bands.After oral administration of 3.BALB/c mice, the serum IgG content of NZ3900 / pNZ8110-LysM-hpaA group was the highest, and that of other recombinant strains was higher than that of negative control group (P0.05N. H. pylori). The rapid urease assay showed that the OD450 value of each recombinant lactic acid bacteria group was lower than that of the control group (P 0.05).Conclusion1.High purity H.pylori recombinant antigen UreBUP Lpp20 HPA Omp22 was obtained.2.All of them were immunoreactive by oral administration.The best effect of immune protection caused by 3.NZ3900/pNZ8110-LysM-hpaA
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392

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