DC靶向适配体修饰的载铜绿假单胞菌DNA疫苗递送系统的构建及体外评价

构建了一种DC靶向适配体修饰的铜绿假单胞菌(Pseudomonas aeruginosa,PA)DNA疫苗递送系统。采用乙醇注入法制备阳离子脂质体,静电吸附法制备载pVAX1-OprF-VP22的阳离子脂质体(Lip-pOprF-VP22),探讨不同DOTAP/pDNA质量比的Lip-pOprF-VP22对pVAX1-OprF-VP22的包封效果、对DC2.4的细胞毒性及转染率,筛选最佳质量比的Lip-pOprF-VP22测定其粒径及Zeta电位;后插法制备DC靶向适配体修饰的载pVAX1-OprF-VP22的阳离子脂质体(Apt-Lip-pOprF-VP22),检测其转染DC2.4后OprF蛋白的表达量及对小鼠骨髓来源树突状细胞(bone marrow-derived dendritic cells,BMDCs)成熟的影响。结果表明,Lip-pOprF-VP22随着DOTAP/pDNA质量比增加包封率逐渐增加,当质量比为5∶1时即能很好的包封pVAX1-OprF-VP22;当Lip-pOprF-VP22作用于DC2.4 24 h或48 h后,不同质量比的Lip-pOprF-VP22对DC2.4的存活率均在80%以上;当DOTAP/pDNA质量比由2∶1增加到10∶1,转染率表现为先增加、后降低的趋势,其中DOTAP/pDNA质量比为4∶1、5∶1时转染率相对较高;当DOTAP/pDNA质量比为5∶1时,Lip-pOprF-VP22粒径为(171.67±1.27)nm,Zeta电位为(11.30±0.57)mV;Apt-Lip-pOprF-VP22转染DC2.4后可表达更多OprF蛋白且可明显促进BMDCs的成熟。     

Association of OprF mutant and disturbance of biofilm and pyocyanin virulence in pseudomonas aeruginosa

Outer membrane porin F (OprF) is a major structural membrane protein of Pseudomonas aeruginosa, a recognised human opportunistic pathogen which is correlated with severe hospital-acquired infections. This study investigating a multiphenotypic approach, based on the comparative study of a wild type strain of P. aeruginosa, its isogenic OprF mutant. Both P. aeruginosa PAO1 and OprF mutant strains were grown in same condition and cultures were subjected to further analysis by SDS PAGE, pyocyanin production and biofilm formation that was analyse using scanning electron microscopy. Based on biofilm formation essay and pyocyanin production, the study showed that OprF plays a dynamic role in P. aeruginosa virulence. The absence of OprF results in slow growth rate corresponded to elongated lag phase and reduced biofilm production also a significance reduction in the production of the quorum-sensing-dependent virulence factors pyocyanin. Accordingly, in the OprF mutant scanning electron microscope “SEM” images showed impaired cellular niche and detached cells when compared to regular attached P. aeruginosa wild type cells in the niche. Taken together, this study shows the contribution of OprF in P. aeruginosa virulence, at least partly through impairment of biofilm, cell to cell attachment in niche and pyocyanin production. This study show a vital link between OprF and virulence factor production, providing novel insights for its role in pathogenicity and future could provide the basis for the development of novel drug targets for antibiotics and vaccines.     

Recombinant N-terminal outer membrane porin (OprF) of Pseudomonas aeruginosa is a promising vaccine candidate against both P. aeruginosa and some strains of Acinetobacter baumannii

Pseudomonas aeruginosa is an evolving pathogen which can cause serious infections especially to immunocompromised patients. Its high resistance profile to antibiotics results in difficulty, and sometimes impossibility, in treating afflicted patients. Developing an effective vaccine against P. aeruginosa is an important approach to tackle this problem. A similar problematic situation exists for Acinetobacter baumannii. Several vaccine candidates have been investigated up till now but still there is no approved vaccine in the market. One important antigen of P. aeruginosa is the outer membrane protein F (OprF) which functions as a porin with relevant important roles in virulence. Previous studies focused mainly on the C-terminal peptidoglycan binding domain of OprF as a vaccine candidate. In the current study, we have investigated the N-terminal porin domain of OprF as a potential vaccine candidate against P. aeruginosa. Histidine-tagged recombinant N-terminal OprF (amino acid range 25–200; OprF_25-200) was overexpressed in Escherichia coli and purified using metal affinity chromatography. Swiss albino mice were immunized with OprF_25-200 adjuvanted with Bacillus Calmette–Guérin (BCG) and alum and the immune response was evaluated. Immunized mice developed antigen-specific IgG1 and IgG2a and were protected against challenge by both P. aeruginosa and a clinical isolate of A. baumannii expressing OprF. Serum from OprF_25-200-immunized mice showed cross-reactivity with both pathogens using western blotting and whole cell enzyme-linked immunosorbent assay (ELISA). To our knowledge, this is the first report to demonstrate that the N-terminal domain of OprF is sufficiently immunogenic to protect against the two pathogens.     

铜绿假单胞菌外膜蛋白OprF基因的克隆与原核表达

[目的]克隆铜绿假单胞菌OprF基因,并进行原核表达,以期进一步开展铜绿假单胞菌基因工程疫苗的研究. [方法]利用PCR技术从铜绿假单胞菌基因组中扩增出OprF基因.采用T-A克隆构建pMD18-T-OprF重组质粒,测序正确后经BamH Ⅰ、Hind Ⅲ双酶切获得OprF片段,插入到表达载体pET32a,获得pET32a-OprF重组表达质粒并在表达宿主菌E.Coli BL21中诱导表达,利用Ni-NTA柱对目的蛋白进行纯化,通过SDS-PAGE电泳和蛋白质印迹进行鉴定. [结果]克隆OprF基因(459 bp)并经DNA测序证实,表达重组蛋白OprF并获得纯化蛋白,经SDS-PAGE电泳和蛋白质印迹鉴定正确.[结论]成功克隆了OprF基因并获得原核表达物,为铜绿假单胞菌的致病性研究和开展基因工程疫苗的研制奠定了基础.     

铜绿假单胞菌重组Bb-OprF疫苗构建及鉴定

目的构建和鉴定铜绿假单胞菌重组双歧杆菌（rBb）-OprF疫苗。方法以铜绿假单胞菌PAOl标准株提取总RNA为模板,自行设计引物,RT-PCR扩增获得OprF抗原编码基因,经酶切、连接定向克隆入大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-OprF。转化E.coliBL21（DE3）感受态细胞,抽提质粒行酶切电泳和测序验证后,电穿孔转化到Bb中,构建铜绿假单胞菌rBb-OprF疫苗,抽提质粒进行PCR扩增鉴定。结果 RT-PCR扩增出1 016bp的OprF编码基因;重组质粒经双酶切鉴定,切出4 947bp的载体片段和10 16bp的目的基因片段;以rBb抽提质粒为模板进行PCR扩增可得到1 016bp的oprF基因片段。结论成功构建了铜绿假单胞菌rBb-OprF疫苗,为该疫苗的进一步研究奠定基础。     

铜绿假单胞菌重组质粒pGEX-OprF的构建及其表达

目的构建并鉴定铜绿假单胞菌重组质粒pGEX-OprF,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法以铜绿假单胞菌PAOl标准株总RNA为模板,自行设计引物,采用RT-PCR方法扩增OprF抗原编码基因,经酶切、连接后定向克隆入大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-OprF,转化E.coliBL21(DE3)感受态细胞,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果 RT-PCR扩增出1 016 bp的OprF编码基因;重组质粒经双酶切和测序鉴定证实OprF基因成功插入pGEX-1λT中;SDS-PAGE分析表达产物分子质量单位约为61 ku,与预期结果一致,表达的蛋白质占菌体总蛋白的16%;Western blot鉴定重组蛋白能被Pa外膜粗抗原免疫小鼠血清识别。结论成功构建了铜绿假单胞菌重组质粒pGEX-OprF,该质粒在E.coliBL21(DE3)中高效融合表达,表达的融合蛋白具有抗原特异性。     

Prospects for oral replicating adenovirus-vectored vaccines

Orally delivered replicating adenovirus (Ad) vaccines have been used for decades to prevent adenovirus serotype 4 and 7 respiratory illness in military recruits, demonstrating exemplary safety and high efficacy. That experience suggests that oral administration of live recombinant Ads (rAds) holds promise for immunization against other infectious diseases, including those that have been refractory to traditional vaccination methods. Live rAds can express intact antigens from free-standing transgenes during replication in infected cells. Alternatively, antigenic epitopes can be displayed on the rAd capsid itself, allowing presentation of the epitope to the immune system both prior to and during replication of the virus. Such capsid-display rAds offer a novel vaccine approach that could be used either independently of or in combination with transgene expression strategies to provide a new tool in the search for protection from infectious disease.     

OprF/I-vaccinated sera inhibit binding of human interferon-gamma to Pseudomonas aeruginosa

The recombinant outer membrane protein OprF/I has been demonstrated in previous studies to protect against Pseudomonas aeruginosa infection through a mechanism of enhanced antibody-mediated opsonophagocytosis. Recent evidence indicates that P. aeruginosa enhances its virulence phenotype as a consequence of binding to human IFN-γ through an outer membrane protein, OprF. In this study, we demonstrate that a single boost injection of OprF/I vaccine elicited a strong OprF/I-specific antibody response in individuals who were previously vaccinated with OprF/I in a clinical trial. The OprF/I-vaccinated sera inhibit P. aeruginosa binding to IFN-γ, suggesting an alternative mechanism by which the OprF/I vaccine confers protection against P. aeruginosa infection.     

铜绿假单胞菌外膜蛋白OprF和OprH基因扩增、融合、克隆及原核表达

目的 在大肠杆菌中串联表达铜绿假单胞菌外膜蛋白OprF和OprH，获得重组融合蛋白OprF／H，为抗铜绿假单胞菌的疫苗研究打下基础。方法 从铜绿假单胞菌中提取基因组DNA，设计PCR引物扩增出OprF和OprH基因，扩增片段经过酶切、连接和PCR扩增后，割胶回收PCR产物并将其克隆至克隆质粒，测序后构建表达质粒pGEX-F／H，并转化大肠杆菌BL21。结果 重组表达质粒经IPTG诱导后表达融合外膜蛋白OprF／H。结论 成功构建融合外膜蛋白OprF／H的原核表达质粒并在大肠杆菌中表达成功。     

Evidence for association of lipopolysaccharide with Pseudomonas fluorescens strain MF0 porin OprF

Lipopolysaccharide (LPS) was found to be associated with the major outer membrane protein OprF of the psychrotrophic bacterium Pseudomonas fluorescens MF0, using two OprF purification procedures. OprF, purified under mild conditions, presented two types of association with LPS: tight (tLPS) and slight (sLPS), both of type R. LPS protected OprF from heat modification and trypsin degradation and facilitated the reincorporation of purified OprF into an artificial lipid bilayer without affecting its pore-forming activity. The size of the OprF channel depended on cell growth temperature, as did the extent of LPS phosphorylation: we suggest that LPS may be involved in modifications of OprF pore formation.