甲型副伤寒沙门氏菌外膜蛋白NmpC、PagC和OmpW作为候选疫苗的初步探讨

发布时间：2018-07-05 00:49

  本文选题：外膜蛋白 + NmpC　； 参考：《南昌大学》2012年硕士论文

【摘要】：甲型副伤寒沙门氏菌临床耐药株的出现使得临床治疗变得十分困难,严重影响包括中国在内的许多发展中国家的人们的健康。目前,我国由于甲型副伤寒疫苗尚未面世,防治形势显得尤其严峻。因此,迫切需要研制并开发出安全、高效、简便、经济的甲型副伤寒疫苗。本论文旨在对甲型副伤寒沙门氏菌外膜蛋白NmpC、 PagC、OmpW进行相关研究,初步探讨其作为候选疫苗的可行性,研究内容综述如下：1、甲型副伤寒沙门氏菌外膜蛋白NmpC与PagC的新型疫苗的初步研究。前期研究表明,NmpC与PagC两个蛋白具有较强的免疫原性和保护力。本文采用宿主-质粒平衡致死系统,成功构建了nmpC*-asd/FWL01、 pagC*-asd/FWL01、nmpC-lpp-ompA/FWL01 和 PpagC-lpp-ompA/FWL01四株重组疫苗株。经鉴定,重组菌株均与宿主菌具有相同的生化特性；质粒稳定性实验证实重组疫苗候选株质粒不会影响宿主菌的生长,能够稳定遗传。重组菌免疫家兔后均有免疫应答反应,ELISA结果显示四株重组菌nmpC*-asd/FWL01、 pagC*-asd/FWL01、nmpC-lpp-ompA/FWL01和pagC-1pp-ompA/F WL01各组特异性抗体滴度分别为6400、6400及12800、6400。2. PagC可溶性抗原串联表达蛋白sPagC的免疫原性研究。以外膜蛋白PagC的氨基酸序列为基础,中间用连结肽连接,构建了重组抗原sPagC的序列。通过全基因合成得到带组氨酸标签的spagC序列,从甲型副伤寒CMCC50973基因组中扩增得到pagC序列,并在5’端加入组氨酸标签。将序列正确的pagC与spagC片段与表达质粒pET-30a连接,转化入表达宿主菌BL21中,构建得到重组表达菌株pagC-pET-30a/BL21和spagC-pET-30a/BL21。诱导重组蛋白大量表达,其中PagC和sPagC分别以包涵体和可溶性的形式表达；经固定化金属离子亲和层析柱纯化,梯度透析使PagC复性,并去除了sPagC中的咪唑,纯度可达90%以上。使用纯化的目的蛋白与氢氧化铝佐剂进行混合后免疫小鼠,通过IgG抗体效价交叉测定对PagC和sPagC的免疫原性进行初步评价及比较。实验结果显示,PagC免疫组小鼠血清IgG抗体的效价较高,而sPagC免疫组则没有明显效价。3、研究ompW在甲型副伤寒中与毒力的相关性。借助λ-Red重组系统成功构建了甲型副伤寒沙门氏菌CMCC 50973基因缺失突变株50973 △ompW::kan。采用低拷贝质粒pACU184,获得了ompW回复互补株。ompW缺失野生株、突变株与互补株的生长曲线基本相同,没有显著差异(P0.05),全自动细菌鉴定仪表明三者的生化性质也几乎完全相同。用ompW野生株、突变株和回复株(1×109 CFU/mL, 1×108CFU/mL, 1×107CFU/mL, 1×106 CFU/mL, 1×105CFU/mL)进行小鼠腹腔注射实验,获得野生株、突变株与回复株的LD50值分别为3.16×106 CFU/mL、2.37×106 CFU/mL,4.22×106 CFU/mL。综上所述,对NmpC、PagC候选疫苗的研究结果表明,四株重组菌均能引起较温和的免疫反应,符合口服活疫苗低免疫原性的特点；PagC可溶性抗原串联表达蛋白sPagC免疫原性较差,可能与引起免疫反应的主要有效成分为疏水性有关；甲型副伤寒沙门氏菌中ompW与毒力无明显相关性,可作为一种潜在的疫苗加以进一步研究。本研究为甲型副伤寒疫苗的后续研究和开发奠定了理论基础。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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