本文選題：外膜蛋白 + NmpC�。� 參考：《南昌大學(xué)》2012年碩士論文

【摘要】：甲型副傷寒沙門氏菌臨床耐藥株的出現(xiàn)使得臨床治療變得十分困難,嚴(yán)重影響包括中國在內(nèi)的許多發(fā)展中國家的人們的健康。目前,我國由于甲型副傷寒疫苗尚未面世,防治形勢顯得尤其嚴(yán)峻。因此,迫切需要研制并開發(fā)出安全、高效、簡便、經(jīng)濟(jì)的甲型副傷寒疫苗。本論文旨在對甲型副傷寒沙門氏菌外膜蛋白NmpC、 PagC、OmpW進(jìn)行相關(guān)研究,初步探討其作為候選疫苗的可行性,研究內(nèi)容綜述如下：1、甲型副傷寒沙門氏菌外膜蛋白NmpC與PagC的新型疫苗的初步研究。前期研究表明,NmpC與PagC兩個蛋白具有較強的免疫原性和保護(hù)力。本文采用宿主-質(zhì)粒平衡致死系統(tǒng),成功構(gòu)建了nmpC*-asd/FWL01、 pagC*-asd/FWL01、nmpC-lpp-ompA/FWL01 和 PpagC-lpp-ompA/FWL01四株重組疫苗株。經(jīng)鑒定,重組菌株均與宿主菌具有相同的生化特性；質(zhì)粒穩(wěn)定性實驗證實重組疫苗候選株質(zhì)粒不會影響宿主菌的生長,能夠穩(wěn)定遺傳。重組菌免疫家兔后均有免疫應(yīng)答反應(yīng),ELISA結(jié)果顯示四株重組菌nmpC*-asd/FWL01、 pagC*-asd/FWL01、nmpC-lpp-ompA/FWL01和pagC-1pp-ompA/F WL01各組特異性抗體滴度分別為6400、6400及12800、6400。2. PagC可溶性抗原串聯(lián)表達(dá)蛋白sPagC的免疫原性研究。以外膜蛋白PagC的氨基酸序列為基礎(chǔ),中間用連結(jié)肽連接,構(gòu)建了重組抗原sPagC的序列。通過全基因合成得到帶組氨酸標(biāo)簽的spagC序列,從甲型副傷寒CMCC50973基因組中擴增得到pagC序列,并在5’端加入組氨酸標(biāo)簽。將序列正確的pagC與spagC片段與表達(dá)質(zhì)粒pET-30a連接,轉(zhuǎn)化入表達(dá)宿主菌BL21中,構(gòu)建得到重組表達(dá)菌株pagC-pET-30a/BL21和spagC-pET-30a/BL21。誘導(dǎo)重組蛋白大量表達(dá),其中PagC和sPagC分別以包涵體和可溶性的形式表達(dá)；經(jīng)固定化金屬離子親和層析柱純化,梯度透析使PagC復(fù)性,并去除了sPagC中的咪唑,純度可達(dá)90%以上。使用純化的目的蛋白與氫氧化鋁佐劑進(jìn)行混合后免疫小鼠,通過IgG抗體效價交叉測定對PagC和sPagC的免疫原性進(jìn)行初步評價及比較。實驗結(jié)果顯示,PagC免疫組小鼠血清IgG抗體的效價較高,而sPagC免疫組則沒有明顯效價。3、研究ompW在甲型副傷寒中與毒力的相關(guān)性。借助λ-Red重組系統(tǒng)成功構(gòu)建了甲型副傷寒沙門氏菌CMCC 50973基因缺失突變株50973 △ompW::kan。采用低拷貝質(zhì)粒pACU184,獲得了ompW回復(fù)互補株。ompW缺失野生株、突變株與互補株的生長曲線基本相同,沒有顯著差異(P0.05),全自動細(xì)菌鑒定儀表明三者的生化性質(zhì)也幾乎完全相同。用ompW野生株、突變株和回復(fù)株(1×109 CFU/mL, 1×108CFU/mL, 1×107CFU/mL, 1×106 CFU/mL, 1×105CFU/mL)進(jìn)行小鼠腹腔注射實驗,獲得野生株、突變株與回復(fù)株的LD50值分別為3.16×106 CFU/mL、2.37×106 CFU/mL,4.22×106 CFU/mL。綜上所述,對NmpC、PagC候選疫苗的研究結(jié)果表明,四株重組菌均能引起較溫和的免疫反應(yīng),符合口服活疫苗低免疫原性的特點；PagC可溶性抗原串聯(lián)表達(dá)蛋白sPagC免疫原性較差,可能與引起免疫反應(yīng)的主要有效成分為疏水性有關(guān)；甲型副傷寒沙門氏菌中ompW與毒力無明顯相關(guān)性,可作為一種潛在的疫苗加以進(jìn)一步研究。本研究為甲型副傷寒疫苗的后續(xù)研究和開發(fā)奠定了理論基礎(chǔ)。
[Abstract]:The emergence of a clinical drug resistant strain of Salmonella paratyphi A has made clinical treatment very difficult and seriously affects the health of many developing countries, including China. At present, the prevention and control situation of a paratyphan Typhoid Vaccine has not yet come out in our country. This paper aims to study the outer membrane protein NmpC, PagC, OmpW of Salmonella paratyphi A, and discuss its feasibility as a candidate vaccine. The contents of the research are as follows: 1, preliminary study on the new vaccine of the outer membrane protein NmpC of Salmonella paratyphi A and PagC. The two proteins of NmpC and PagC have strong immunogenicity and protective ability. In this paper, four recombinant vaccine strains of nmpC*-asd/FWL01, pagC*-asd/FWL01, nmpC-lpp-ompA/FWL01 and PpagC-lpp-ompA/FWL01 were successfully constructed by using the host plasmid balance lethal system. The recombinant strains were identified as the same biochemical characteristics with the host bacteria; The stability test confirmed that the plasmid of the recombinant vaccine did not affect the growth of the host bacteria and could stabilize the inheritance. The recombinant strain had immune response after the immunization of the rabbit. The results of ELISA showed that four recombinant strains of nmpC*-asd/FWL01, pagC*-asd/FWL01, nmpC-lpp-ompA/FWL01 and pagC-1pp-ompA /F WL01 were respectively the specific antibody titers of 64006400 groups, respectively. The immunogenicity of the 128006400.2. PagC soluble antigen series expression protein sPagC was studied. Based on the amino acid sequence of the outer membrane protein PagC, the sequence of the recombinant antigen sPagC was constructed in the middle. The spagC sequence with the histidine tag was synthesized through the whole gene synthesis, and amplified from the paratyphi A CMCC50973 genome. To the pagC sequence and add the histidine label at the 5 'end, the correct pagC and spagC fragment are connected with the expression plasmid pET-30a to express the host bacteria BL21, and the recombinant expression strain pagC-pET-30a/BL21 and spagC-pET-30a/BL21. are constructed to induce a large amount of recombinant protein expression, in which PagC and sPagC are inclusion bodies and soluble proteins respectively. It was expressed in form, purified by immobilized metal ion affinity chromatography column, refolding PagC and removing imidazole in sPagC, with the purity of more than 90%. The purified target protein was mixed with aluminum hydroxide adjuvant to immunize mice, and the immunogenicity of PagC and sPagC was evaluated by IgG antibody titer, and the immunogenicity of PagC and sPagC was preliminarily evaluated and The experimental results showed that the titer of serum IgG antibody in the PagC immunization group was higher, while the sPagC immune group had no significant titer.3, and the correlation between ompW and virulence in paratyphoid A was studied. With the aid of the lambda -Red recombinant system, the CMCC 50973 based deletion mutant of Salmonella paratyphi was successfully constructed and the low torture was used in kan.. Bainite plasmid pACU184, obtained the ompW recovery complementary strain.OmpW deletion wild strain, the growth curve of the mutants and the complementary strains were basically the same, there was no significant difference (P0.05). The biochemical properties of the three were almost the same. With ompW wild strain, mutant and recovery strain (1 * 109 CFU/mL, 1 * 108CFU/mL, 1 * 107CFU/mL, 1 *). 106 CFU/mL, 1 x 105CFU/mL) mice intraperitoneal injection experiment, the wild strain, the LD50 value of the mutant strain and the response strain were 3.16 x 106 CFU/mL, 2.37 x 106 CFU/mL, 4.22 x 106 CFU/mL., the results of NmpC, PagC candidate vaccine research showed that four recombinant bacteria could cause a mild immune response, in line with the low oral live vaccine The characteristics of immunogenicity; the poor immunogenicity of the PagC soluble antigen tandem expression protein sPagC may be related to the hydrophobicity of the main effective component of the immune response; the ompW in Salmonella paratyphi A has no significant correlation with the virulence. It can be used as a potential vaccine plus further study. This study is a paratyphoid disease. The follow-up study and development of the seedlings lay a theoretical foundation.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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相關(guān)期刊論文 前1條

1 王景;穆媛媛;黎庶;陳志瑾;熊坤;叢延廣;;革蘭陰性菌基因敲除載體的構(gòu)建及其應(yīng)用[J];第三軍醫(yī)大學(xué)學(xué)報;2009年23期

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本文編號：2098189