efficient-transfection-vitro-transcribed-mrna-cultured-cells-using

Efficient Transfection of <em>In vitro</em> Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

Third Military Medical University

To express a fusion protein of Neisseria gonorrhoeae with a mucosal adjuvant.

[Fusion expression of Neisseria gonorrhoeae outmembrane protein with a mucosal adjuvant].

Interleukin-24 (IL-24) can induce apoptosis of a broad range of tumor cells, and this function of IL-24 is independent of classic tumor suppressor genes, such as p53, Rb and p16. Here, we report the expression, purification and preparation of a recombinant IL-24 protein (rIL-24) without post-translational modifications, which may selectively induce apoptosis of tumor cells in vitro. We found that non-fusion rIL-24 was not able to be expressed by vectors pET11c, 28a, and 22b in Escherichia coli. To obtain recombinant non-fusion IL-24 protein, the encoding region for IL-24 was cloned between KpnI and BamHI in pET32a. The Trx (Thioredoxin)/IL-24 fusion proteins were expressed in the form of inclusion bodies in E. coli host strain BL21 (DE21). The expression level was more than 30% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >96% were obtained. Trx/IL-24 proteins were digested by enterokinase (EK) to both Trx and rIL-24 fragments which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rIL-24 (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the rIL-24 may have cancer therapeutic applications.

Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli.

&#x94C1;&#x88AB;&#x516C;&#x8BA4;&#x7684;&#x91CD;&#x8981;&#x5FAE;&#x91CF;&#x5143;&#x7D20;&#xFF0C;&#x5927;&#x591A;&#x6570;&#x751F;&#x7269;&#x5305;&#x62EC;&#x81F4;&#x75C5;&#x6027;&#x7EC6;&#x83CC;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;HugZ&#xFF0C;&#x4E00;&#x79CD;&#x86CB;&#x767D;&#x4E0E;&#x8840;&#x7EA2;&#x7D20;&#x94C1;&#x5229;&#x7528;&#x7387;&#xFF0C;&#x6709;&#x5173;&#x7684;&#x53C2;&#x4E0E;&#x4ECE;&#x4E3B;&#x673A;&#x94C1;&#x7EC6;&#x83CC;&#x8D2D;&#x7F6E;&#x3002;&#x6211;&#x4EEC;&#x4EE5;&#x524D;&#x6240;&#x6307;&#x51FA; hugZ &#x540C;&#x7CFB;&#x7269;&#x81EA;&#x9002;&#x5E94;&#x7684;&#x5E7D;&#x95E8;&#x87BA;&#x6746;&#x83CC; (&#x5E7D;&#x95E8;) &#x6B96;&#x6C11;&#x5316;&#x4E0E;&#x76F8;&#x5173;&#x4E3B;&#x8981;&#x7684;&#x80C3;&#x80A0;&#x9053;&#x81F4;&#x75C5;&#x83CC;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x5176;&#x786E;&#x5207;&#x7684;&#x751F;&#x7406;&#x4F5C;&#x7528;&#x4ECD;&#x7136;&#x4E0D;&#x6E05;&#x695A;&#x3002;

HugZ&#xFF0C;&#x4ECE;&#x5E7D;&#x95E8;&#x87BA;&#x6746;&#x83CC;&#x7684;&#x8840;&#x7EA2;&#x7D20;&#x52A0;&#x6C27;&#x9176;&#x7684;&#x529F;&#x80FD;&#x9274;&#x5B9A;&#x3002;

Iron is recognized as an important trace element, essential for most organisms including pathogenic bacteria. HugZ, a protein related to heme iron utilization, is involved in bacterial acquisition of iron from the host. We previously observed that a hugZ homologue is correlated with the adaptive colonization of Helicobacter pylori (H. pylori), a major gastro-enteric pathogen. However, its exact physiological role remains unclear.

Functional identification of HugZ, a heme oxygenase from Helicobacter pylori.

Fer est reconnu comme un oligo-&eacute;l&eacute;ment important, essentiel pour la plupart des organismes dont les bact&eacute;ries pathog&egrave;nes. HugZ, une prot&eacute;ine li&eacute;e &agrave; l&#39;utilisation de fer h&eacute;mique, est impliqu&eacute; dans l&#39;acquisition bact&eacute;rienne de fer &agrave; partir de l&#39;h&ocirc;te. Nous avons d&eacute;j&agrave; observ&eacute; qu&#39;un homologue hugZ est corr&eacute;l&eacute; avec la colonisation adaptative de Helicobacter pylori (H. pylori), un pathog&egrave;ne majeur gastro-intestinale. Cependant, son r&ocirc;le physiologique exacte reste incertaine.

Identification fonctionnelle de HugZ, une h&egrave;me oxyg&eacute;nase de Helicobacter pylori.

Eisen wird als eine wichtige Spurenelement, notwendig f&uuml;r die meisten Organismen einschlie&szlig;lich pathogene Bakterien erkannt. HugZ, ein Protein mit Bezug zu H&auml;m-Eisen-Auslastung, ist bakterielle Erwerb von Eisen aus dem Host beteiligt. Wir zuvor festgestellt, dass ein HugZ Homolog ist korreliert mit der adaptiven Kolonisierung des Helicobacter Pylori (H. Pylori), eines bedeutenden Gastro-enterale Erregers. Seine genaue physiologische Rolle bleibt jedoch unklar.

Funktionale Identifikation von HugZ, ein H&auml;m-Oxygenase von Helicobacter Pylori.

Ferro &egrave; riconosciuta come un importante elemento traccia, essenziale per la maggior parte degli organismi compreso batteri patogeni. HugZ, una proteina relative all&#39;utilizzazione di ferro eme, &egrave; coinvolto nell&#39;acquisizione batterica del ferro dall&#39;host. Abbiamo precedentemente osservato che un omologo hugZ &egrave; correlato con la colonizzazione adattiva di Helicobacter pylori (H. pylori), un importante agente patogeno gastro-enterico. Tuttavia, il suo esatto ruolo fisiologico rimane poco chiaro.

Identificazione funzionale di HugZ, un eme ossigenasi da Helicobacter pylori.

&#x9244;&#x75C5;&#x539F;&#x7D30;&#x83CC;&#x3092;&#x542B;&#x3080;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x751F;&#x7269;&#x306E;&#x305F;&#x3081;&#x91CD;&#x8981;&#x306A;&#x91CD;&#x8981;&#x306A;&#x30C8;&#x30EC;&#x30FC;&#x30B9;&#x8981;&#x7D20;&#x3068;&#x3057;&#x3066;&#x8A8D;&#x8B58;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;HugZ&#x3001;&#x30D8;&#x30E0;&#x9244;&#x306E;&#x4F7F;&#x7528;&#x7387;&#x306B;&#x95A2;&#x9023;&#x3059;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306F;&#x9244;&#x3001;&#x30DB;&#x30B9;&#x30C8;&#x304B;&#x3089;&#x306E;&#x7D30;&#x83CC;&#x306E;&#x8CB7;&#x53CE;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x4EE5;&#x524D; hugZ &#x30DB;&#x30E2;&#x30ED;&#x30B0; &#x30D4;&#x30ED;&#x30EA;&#x83CC; &#xFF08;&#x30D8;&#x30EA;&#x30B3;&#x30D0;&#x30AF;&#x30BF;&#x30FC;&#x30D4;&#x30ED;&#x30EA;&#xFF09; &#x306E;&#x9069;&#x5FDC;&#x306E;&#x690D;&#x6C11;&#x5730;&#x5316;&#x3068;&#x76F8;&#x95A2;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x89B3;&#x5BDF;&#x4E3B;&#x8981;&#x306A;&#x80C3;&#x8178;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x305D;&#x306E;&#x6B63;&#x78BA;&#x306A;&#x751F;&#x7406;&#x7684;&#x5F79;&#x5272;&#x306F;&#x4E0D;&#x660E;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

HugZ&#x3001;&#x30D4;&#x30ED;&#x30EA;&#x83CC;&#x30D0;&#x30AF;&#x30C6;&#x30EA;&#x30A2;&#x7531;&#x6765;&#x30D8;&#x30E0;&#x30AA;&#x30AD;&#x30B7;&#x30B2;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x6A5F;&#x80FD;&#x7684;&#x540C;&#x5B9A;&#x3002;

&#xC911;&#xC694; &#xD55C; &#xCD94;&#xC801; &#xC694;&#xC18C;, &#xBCD1;&#xC6D0; &#xC131; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xB97C; &#xD3EC;&#xD568; &#xD55C; &#xB300;&#xBD80;&#xBD84;&#xC758; &#xC0DD;&#xBB3C;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778; &#xCCA0; &#xC778;&#xC2DD; &#xB429;&#xB2C8;&#xB2E4;. HugZ, heme &#xCCA0; &#xD65C;&#xC6A9;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xB2E8;&#xBC31;&#xC9C8;&#xC740; &#xD638;&#xC2A4;&#xD2B8;&#xC5D0;&#xC11C; &#xCCA0;&#xC758; &#xC138;&#xADE0;&#xC131; &#xC778;&#xC218;&#xC5D0; &#xAD00;&#xC5EC;. &#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xC804;&#xC5D0; hugZ homologue &#xD5EC;&#xB9AC;&#xCF54;&#xBC15;&#xD130; &#xD30C;&#xC77C;&#xB85C;&#xB9AC; (&#xD5EC;&#xB9AC;&#xCF54;&#xBC15;&#xD130; &#xD30C;&#xC77C;&#xB85C;&#xB9AC;), &#xC801;&#xC751;&#xD615; &#xC2DD;&#xBBFC;&#xC9C0;&#xC640; &#xC0C1;&#xAD00;&#xC740; &#xAD00;&#xCC30; &#xC8FC;&#xC694; gastro-&#xC7A5;&#xC758; &#xBCD1;&#xC6D0; &#xCCB4;. &#xADF8;&#xB7EC;&#xB098;, &#xADF8;&#xAC83;&#xC758; &#xC815;&#xD655;&#xD55C; &#xC0DD;&#xB9AC; &#xC801; &#xC5ED;&#xD560; &#xBD88;&#xBD84;&#xBA85; &#xB0A8;&#xC544; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

HugZ, &#xD5EC;&#xB9AC;&#xCF54;&#xBC15;&#xD130; &#xD30C;&#xC77C;&#xB85C;&#xB9AC;&#xC5D0;&#xC11C; heme oxygenase&#xC758; &#xAE30;&#xB2A5; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

Hierro es reconocido como un oligoelemento importante, esencial para la mayor&iacute;a de los organismos incluyendo bacterias pat&oacute;genas. HugZ, una prote&iacute;na relacionada con la utilizaci&oacute;n de hierro hemo, est&aacute; involucrado en la adquisici&oacute;n bacteriana del hierro del anfitri&oacute;n. Anteriormente, observamos que un hom&oacute;logo hugZ est&aacute; correlacionado con la adaptaci&oacute;n colonizaci&oacute;n de Helicobacter pylori (H. pylori), un importante pat&oacute;geno gastroent&eacute;rica. Sin embargo, su papel fisiol&oacute;gico exacto sigue siendo confuso.

Identificaci&oacute;n funcional de HugZ, un heme oxigenasa de Helicobacter pylori.

Shiga toxins consist of enzymatically active A and B subunit multimers. The A subunit of shiga-like toxins can be proteolytically cleaved into two parts, A(1) and A(2), with A(1) being responsible for toxic activity. Antibody neutralizing the A(1) subunit of shiga toxin may protect against infection of the enterohemorrhagic Escherichia coli (EHEC O157:H7). It was difficult to express the full-length A(1) subunit of shiga toxin 2 (stx2A(1)) in a previous study. We have now analyzed the full-length of stx2A(1) using bioinformatics software. The data show that the carboxyl terminal (of ~15 amino-acid residues) has strong hydrophobicity and low antigenicity. We cloned and expressed a truncated fragment of stx2A(1) (15 amino-acid residues of the carboxyl terminal being removed), designated stx2a(1), which can evoke a humoral immune response. Anti-Stx2a(1) antibodies can neutralize the native shiga toxin 2 both in vivo and in vitro, which suggests that Stx2a(1) serves as a candidate immunogen for a subunit vaccine that can also be used as the antigen to screen phage anti-shiga toxin antibody libraries.

Cloning a truncated fragment (stx2a(1)) of the shiga-like toxin 2A (1) subunit of EHEC O157:H7: candidate immunogen for a subunit vaccine.

Enterohemorrhagic Escherichia coli (EHEC) causes a wide range of systematic diseases in human and animals in 2 main ways: (1) production of Shiga toxin (Stx) and (2) induction of actin polymerization characterized by attaching and effacing (A/E) lesions. Stx is commonly targeted in the development of drugs and vaccines to control EHEC infection for its indispensible contribution to EHEC pathogenesis. In this study, we isolated a Stx-producing EHEC O157:H7 isolate 00B015 and found that its ability to induce actin polymerization was impaired. In addition, it reduces pathogenicity and decreases mortality in mice. Our results report a Stx-producing but virulence-attenuated EHEC isolate 00B015 and suggest that the formation of actin polymerization may help Stx-induced pathogenesis and have a more important contribution in EHEC infections.

Characterization of enterohemorrhagic Escherichia coli O157:H7 00B015: a Shiga toxin producing but virulence-attenuated isolate.

Mutants of pertussis toxin (PT) S1 subunit and filamentous hemagglutinin (FHA) type I immunodominant domain from Bordetella pertussis (B. pertussis) are considered to be effective candidate antigens for acellular pertussis vaccines; however, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. In this paper, the gene sequences of a S1 mutant C180-R9K/E129G (mS1) and a truncated peptide named Fs from FHA type I immunodominant domain were linked together and constructed to pET22b expression vector as a fusion gene; after inducing with IPTG, it was highly expressed in E. coli BL21 (DE3) as inclusion body. The fusion protein FsmS1 was purified from cell lysates and refolded successfully. The result of Western blotting indicate that it was able to react with both anti-S1 and anti-FHA McAbs; antiserum produced from New Zealand white rabbits immunized with this protein was able to recognize both native PT and FHA antigens as determined by western blotting. These data have provided a novel feasible method to produce PT S1 subunit and FHA type I immunodominant domain in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against B. pertussis infection.

Fusion expression and immunogenicity of Bordetella pertussis PTS1-FHA protein: implications for the vaccine development.

&#x7834;&#x4F24;&#x98CE;&#x3001; &#x4E25;&#x91CD;&#x4F20;&#x67D3;&#x6027;&#x75BE;&#x75C5;&#xFF0C;&#x88AB;&#x5F15;&#x8D77;&#x7834;&#x4F24;&#x98CE;&#x6BD2;&#x7D20; (TT) &#x4ECE;&#x68AD;&#xFF0C;&#x4ECD;&#x7136;&#x662F;&#x5C3D;&#x7BA1;&#x9884;&#x9632;&#x6218;&#x7565;&#x6700;&#x91CD;&#x8981;&#x7684;&#x672A;&#x89E3;&#x51B3;&#x7684;&#x5065;&#x5EB7;&#x95EE;&#x9898;&#x4E4B;&#x4E00;&#x3002;&#x7FA7;&#x57FA;&#x7EC8;&#x7AEF;&#x7684; TT (TTC) &#x662F;&#x8D1F;&#x8D23;&#x5BF9;&#x795E;&#x7ECF;&#x5143;&#x7684; TT &#x7ED1;&#x5B9A;&#xFF0C;&#x5176;&#x6BD2;&#x6027;&#x548C;&#x5DF2;&#x88AB;&#x8BC1;&#x660E;&#x662F;&#x514D;&#x75AB;&#x539F;&#x6027;&#x548C;&#x4FDD;&#x62A4;&#x6027;&#x7684;&#x5404;&#x79CD;&#x5F62;&#x5F0F;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6781;&#x4E3A;&#x6709;&#x8DA3;&#xFF0C;&#x4EE5;&#x8BC6;&#x522B;&#x6297;&#x539F;&#x8868;&#x4F4D; TTC &#x5728;&#x5206;&#x5B50;&#x6C34;&#x5E73;&#x4E0A;&#x3002;&#x5728;&#x6B64;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x751F;&#x6210;&#x548C;&#x5355;&#x514B;&#x9686;&#x6297;&#x4F53;&#xFF0C;5 4&#xFF0C;&#x6291;&#x5236; TT &#x7ED1;&#x5B9A;&#x5230;&#x5176;&#x53D7;&#x4F53;&#xFF0C;&#x5E76;&#x6709;&#x6548;&#x5730;&#x4FDD;&#x62A4;&#x5728; 73.7 IU/mg&#x3002;&#x6B64;&#x5916;&#xFF0C;5 4 &#x627F;&#x8BA4;&#x65B0;&#x578B;&#x7EBF;&#x6027;&#x6297;&#x539F;&#x8868;&#x4F4D;&#x4E0A; TT&#xFF0C;&#x5373; TC((1155-1171)) &#x8DE8;&#x8D8A;&#x4ECE; Lys1155 &#x5230; Val1171&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6240;&#x793A; TC((1155-1171)) &#x4E89;&#x53D6;&#x4FDD;&#x62A4;&#x5C0F;&#x9F20;&#x5BF9;&#x6311;&#x6218;&#x4E0E; TT &#x7684; IgG &#x8840;&#x6E05;&#x7684;&#x751F;&#x4EA7;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E;&#xFF0C;TC((1155-1171)) &#x548C;&#x5355;&#x514B;&#x9686;&#x6297;&#x4F53; 5 C 4 &#x662F;&#x5F88;&#x597D;&#x7684;&#x5019;&#x9009;&#x6297;&#x539F;&#x8868;&#x4F4D;&#x4E3A;&#x57FA;&#x7840;&#x7684;&#x75AB;&#x82D7;&#x548C;&#x9884;&#x9632;&#x7834;&#x4F24;&#x98CE;&#x7684;&#x75AB;&#x82D7;&#x6CBB;&#x7597;&#x6027;&#x6297;&#x4F53;&#x7684;&#x53D1;&#x5C55;&#x3002;

&#x5728;&#x4FDD;&#x62A4;&#x7684;&#x5355;&#x514B;&#x9686;&#x6297;&#x4F53;&#x6240;&#x627F;&#x8BA4;&#x7684;&#x7834;&#x4F24;&#x98CE;&#x6BD2;&#x7D20;&#x65B0;&#x578B;&#x7EBF;&#x6027;&#x6297;&#x539F;&#x8868;&#x4F4D;&#x7684;&#x9274;&#x5B9A;&#xFF1A; &#x75AB;&#x82D7;&#x8BBE;&#x8BA1;&#x7684;&#x5F71;&#x54CD;&#x3002;

Tetanus, a severe infectious disease, is caused by tetanus toxin (TT) from Clostridium tetani, which remains one of the most critical unsolved health problems despite preventive strategies. The carboxyl terminal of TT (TTC) is responsible for the binding of TT to neurons and for its toxicity and has been proven to be immunogenic and protective in various forms. It would therefore be extremely interesting to identify the epitope on TTC at a molecular level. In this study, we generated a neutralizing monoclonal antibody, 5C4, which inhibited TT binding to its receptor and was efficiently protective at 73.7 IU/mg. Moreover, 5C4 recognized a novel linear epitope on TT, namely TC((1155-1171)), which spans from Lys1155 to Val1171. In addition, TC((1155-1171)) was shown to elicit the production of a serum IgG that protected mice against a challenge with TT. These results suggested that TC((1155-1171)) and the monoclonal antibody 5C4 are good candidates for the development of epitope-based vaccines and therapeutic antibodies against tetanus.

Identification of a novel linear epitope in tetanus toxin recognized by a protective monoclonal antibody: implications for vaccine design.

T&eacute;tanos, une maladie infectieuse grave, est caus&eacute; par la toxine t&eacute;tanique (TT) de Clostridium tetani, qui reste l&#39;un des probl&egrave;mes plus critiques de sant&eacute; non r&eacute;solu malgr&eacute; les strat&eacute;gies pr&eacute;ventives. Le terminal de carboxyle de TT (TTC) est responsable pour la liaison du TT aux neurones et de sa toxicit&eacute; et s&#39;est av&eacute;r&eacute; pour &ecirc;tre immunog&egrave;nes et protection sous diverses formes. Il serait donc tr&egrave;s int&eacute;ressant identifier l&#39;&eacute;pitope sur TTC au niveau mol&eacute;culaire. Dans cette &eacute;tude, nous avons g&eacute;n&eacute;r&eacute; un neutralisant anticorps monoclonal, 5 4, qui inhibe la liaison TT &agrave; son r&eacute;cepteur et est efficacement protection &agrave; 73,7 UI/mg. En outre, 5 4 reconna&icirc;tre un &eacute;pitope lin&eacute;aire roman sur TT, nomm&eacute;ment TC((1155-1171)), qui s&#39;&eacute;tend de Lys1155 &agrave; Val1171. En outre, TC((1155-1171)) s&#39;est av&eacute;r&eacute; d&eacute;clenchent la production d&#39;un s&eacute;rum IgG qui prot&eacute;geait les souris contre un d&eacute;fi avec TT. Ces r&eacute;sultats sugg&egrave;rent que TC((1155-1171)) et l&#39;anticorps monoclonal 5C 4 sont de bons candidats pour le d&eacute;veloppement de vaccins &eacute;pitopes et les anticorps th&eacute;rapeutiques contre le t&eacute;tanos.

Identification d&#39;un &eacute;pitope lin&eacute;aire roman en toxine t&eacute;tanique reconnue par un anticorps monoclonaux protecteurs : implications pour la conception de vaccins.

Tetanus, eine schwere Infektionskrankheit, verursacht durch Tetanus-Toxin (TT) von Clostridium Tetani, das bleibt eines der wichtigsten ungel&ouml;sten Gesundheitsprobleme trotz pr&auml;ventive Strategien. Das Karboxyl-Terminal von TT (TTC) ist verantwortlich f&uuml;r die Bindung des TT zu Neuronen und seine Toxizit&auml;t und ist nachgewiesen worden, immunogen und sch&uuml;tzende in verschiedenen Formen sein. Es w&auml;re daher sehr interessant, die Epitope auf TTC auf molekularer Ebene zu identifizieren. In dieser Studie haben wir einen neutralisierende monoklonale Antik&ouml;rper, 5 4, die TT-Bindung an seinen Rezeptor gehemmt und war effizient sch&uuml;tzende bei 73.7 IU/mg generiert. Dar&uuml;ber hinaus erkannt 5 4 eine neuartige lineare Epitope auf TT, n&auml;mlich die TC((1155-1171)), die von Lys1155 bis Val1171 erstreckt. Dar&uuml;ber hinaus wurde die Produktion von einem Serum IgG zu entlocken, die M&auml;use gegen eine Herausforderung mit TT gesch&uuml;tzt TC((1155-1171)) gezeigt. Diese Ergebnisse vorgeschlagen, die TC((1155-1171)) und der monoklonale Antik&ouml;rper 5 C 4 sind gute Kandidaten f&uuml;r die Entwicklung von Impfstoffen Epitop-basierte und therapeutische Antik&ouml;rper gegen Tetanus.

Identifizierung der eine neuartige lineare Epitope in Tetanus-Toxin von einem sch&uuml;tzenden monoklonalen Antik&ouml;rper erkannt: Implikationen f&uuml;r Impfstoff-Design.

Tetano, una grave malattia infettiva, &egrave; causata dalla tossina tetanica (TT) dal Clostridium tetani, che rimane uno dei problemi pi&ugrave; critici di salute irrisolti nonostante strategie preventive. Il terminale carbossilico di TT (TTC) &egrave; responsabile per l&#39;associazione di TT ai neuroni e per la sua tossicit&agrave; e ha dimostrato di essere immunogenico e protettivo in varie forme. Quindi sarebbe estremamente interessante identificare l&#39;epitopo su TTC a livello molecolare. In questo studio, abbiamo generato un neutralizzante anticorpo monoclonale, 4 5, che inibita associazione TT al suo recettore e fu efficacemente protettivo a IU 73.7/mg. Inoltre, 5 4 riconosciuto un romanzo epitopo lineare su TT, vale a dire TC((1155-1171)), che si estende da Lys1155 a Val1171. Inoltre, TC((1155-1171)) ha dimostrato di provocare la produzione di un siero IgG che proteggevano i topi contro una sfida con TT. Questi risultati suggeriscono che TC((1155-1171)) e l&#39;anticorpo monoclonale 5 C 4 sono buoni candidati per lo sviluppo di vaccini basati su epitopo e terapeutici anticorpi contro il tetano.

Identificazione di un romanzo epitopo lineare in tossina tetanica riconosciuto da un anticorpo monoclonale protettivo: implicazioni per la progettazione di vaccini.

&#x7834;&#x50B7;&#x98A8;&#x3001;&#x6DF1;&#x523B;&#x306A;&#x4F1D;&#x67D3;&#x75C5;&#x306E;&#x4E88;&#x9632;&#x6226;&#x7565;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x6700;&#x3082;&#x91CD;&#x8981;&#x306A;&#x672A;&#x89E3;&#x6C7A;&#x306E;&#x5065;&#x5EB7;&#x554F;&#x984C;&#x306E; 1 &#x3064;&#x306E;&#x307E;&#x307E;&#x7834;&#x50B7;&#x98A8;&#x304B;&#x3089;&#x7834;&#x50B7;&#x98A8;&#x6BD2;&#x7D20; &#xFF08;TT&#xFF09; &#x304C;&#x539F;&#x56E0;&#x3067;&#x3059;&#x3002;&#x30AB;&#x30EB;&#x30DC;&#x30AD;&#x30B7;&#x30EB;&#x57FA;&#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB; TT &#xFF08;TTC&#xFF09; &#x306F; TT &#x306E;&#x7D50;&#x5408;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x305D;&#x306E;&#x6BD2;&#x6027;&#x306B;&#x62C5;&#x5F53;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3001;&#x514D;&#x75AB;&#x539F;&#x6027;&#x3068;&#x69D8;&#x3005; &#x306A;&#x5F62;&#x3067;&#x4FDD;&#x8B77;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x5B9F;&#x8A3C;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;TTC &#x3092;&#x5206;&#x5B50;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x306E;&#x30A8;&#x30D4;&#x30C8;&#x30FC;&#x30D7;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x306F;&#x8208;&#x5473;&#x6DF1;&#x3044;&#x3067;&#x3057;&#x3087;&#x3046;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;, &#x4E2D;&#x548C;&#x30E2;&#x30CE;&#x30AF;&#x30ED;&#x30FC;&#x30CA;&#x30EB; TT &#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x305D;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x963B;&#x5BB3;&#x3057;&#x3001;73.7 IU/mg &#x3067;&#x52B9;&#x7387;&#x7684;&#x306B;&#x4FDD;&#x8B77;&#x3055;&#x308C;&#x305F;&#x6297;&#x4F53;&#x3001;5 4 &#x3092;&#x751F;&#x6210;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x307E;&#x305F;&#x3001;5 4 &#x5C0F;&#x8AAC;&#x7DDA;&#x5F62;&#x30A8;&#x30D4;&#x30C8;&#x30FC;&#x30D7; TT&#x3001;Lys1155 &#x304B;&#x3089; Val1171 &#x306B;&#x307E;&#x305F;&#x304C;&#x308B; TC((1155-1171)) &#x3067;&#x3059;&#x306A;&#x308F;&#x3061;&#x8A8D;&#x8B58;&#x3002;&#x307E;&#x305F;&#x3001;TC((1155-1171)) &#x30DE;&#x30A6;&#x30B9; TT &#x3068;&#x30C1;&#x30E3;&#x30EC;&#x30F3;&#x30B8;&#x306B;&#x5BFE;&#x3057;&#x3066;&#x4FDD;&#x8B77;&#x3055;&#x308C;&#x305F; IgG &#x8840;&#x6E05;&#x306E;&#x751F;&#x7523;&#x3092;&#x5F15;&#x304D;&#x51FA;&#x3059;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;TC((1155-1171)) &#x3068;&#x30E2;&#x30CE;&#x30AF;&#x30ED;&#x30FC;&#x30CA;&#x30EB;&#x6297;&#x4F53; 5 C 4 &#x30A8;&#x30D4;&#x30C8;&#x30FC;&#x30D7;&#x57FA;&#x3065;&#x304B;&#x305B;&#x3066;&#x3044;&#x305F;&#x30EF;&#x30AF;&#x30C1;&#x30F3;&#x304A;&#x3088;&#x3073;&#x6CBB;&#x7642;&#x7528;&#x6297;&#x4F53;&#x3092;&#x7834;&#x50B7;&#x98A8;&#x306E;&#x958B;&#x767A;&#x306E;&#x305F;&#x3081;&#x306E;&#x826F;&#x3044;&#x5019;&#x88DC;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

&#x5C0F;&#x8AAC;&#x306E;&#x7DDA;&#x5F62;&#x30A8;&#x30D4;&#x30C8;&#x30FC;&#x30D7;&#x7834;&#x50B7;&#x98A8;&#x6BD2;&#x7D20;&#x306B;&#x3088;&#x308B;&#x4FDD;&#x8B77;&#x30E2;&#x30CE;&#x30AF;&#x30ED;&#x30FC;&#x30CA;&#x30EB;&#x6297;&#x4F53;&#x306E;&#x8A8D;&#x8B58;&#x3067;&#x306E;&#x540C;&#x5B9A;&#xFF1A; &#x30EF;&#x30AF;&#x30C1;&#x30F3;&#x8A2D;&#x8A08;&#x306E;&#x305F;&#x3081;&#x306E;&#x542B;&#x610F;&#x3002;

&#xD30C;&#xC0C1;&#xD48D;, &#xC2EC;&#xAC01;&#xD55C; &#xC804;&#xC5FC; &#xC131; &#xC9C8;&#xBCD1; &#xC608;&#xBC29; &#xC804;&#xB7B5;&#xC5D0;&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0; &#xAC00;&#xC7A5; &#xC911;&#xC694; &#xD55C; &#xBBF8;&#xD574;&#xACB0;&#xB41C; &#xAC74;&#xAC15; &#xBB38;&#xC81C;&#xC758; &#xD558;&#xB098; Clostridium tetani&#xC5D0;&#xC11C; &#xD30C;&#xC0C1;&#xD48D; &#xB3C5; &#xC18C; (TT)&#xC5D0; &#xC758;&#xD574; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;. Carboxyl &#xD130;&#xBBF8;&#xB110; TT (TTC)&#xC758; &#xB274;&#xB7F0;&#xC5D0; TT&#xC758; &#xBC14;&#xC778;&#xB529; &#xBC0F; &#xADF8;&#xAC83;&#xC758; &#xB3C5;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xCC45;&#xC784;&#xC740; immunogenic &#xBC0F; &#xB2E4;&#xC591; &#xD55C; &#xD615;&#xD0DC;&#xB85C; &#xBCF4;&#xD638; &#xB420; &#xC785;&#xC99D; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xAC83;&#xC740; &#xB530;&#xB77C;&#xC11C; &#xC2DD;&#xBCC4; &#xBD84;&#xC790; &#xC218;&#xC900; TTC&#xC5D0; &#xD53C;&#xD1A0; &#xD504;&#xB97C; &#xB9E4;&#xC6B0; &#xD765;&#xBBF8;&#xB85C;&#xC6B4; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xC911;&#xD654; &#xD074;&#xB860; &#xD56D; &#xCCB4;, 5C 4 TT &#xBC14;&#xC778;&#xB529; &#xADF8;&#xAC83;&#xC758; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xC5B5;&#xC81C; &#xD558; &#xACE0;&#xB294; 73.7 IU/mg&#xC5D0; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xBCF4;&#xD638; &#xD558;&#xB294; &#xC0DD;&#xC131; &#xB429;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, 5C 4 TT, &#xC989; TC((1155-1171)), lys1155&#xC5D0;&#xC11C; val1171&#xC5D0; &#xAC78;&#xCCD0; &#xC18C;&#xC124; &#xC120;&#xD615; epitope &#xC778;&#xC2DD;. &#xB610;&#xD55C;, TC((1155-1171)) &#xC720;&#xB3C4; &#xD608; &#xCCAD; IgG TT&#xC640;&#xB3C4; &#xC804;&#xC5D0; &#xB300; &#xD55C; &#xC950;&#xB97C; &#xBCF4;&#xD638; &#xD558;&#xB294; &#xC0DD;&#xC0B0;&#xC744; &#xD45C;&#xC2DC; &#xD588;&#xB2E4;. &#xC774; &#xACB0;&#xACFC; &#xC81C;&#xC548; &#xADF8; TC((1155-1171)) &#xBC0F; &#xB2E8;&#xC77C; &#xD074;&#xB860; &#xD56D; &#xCCB4; 5 C 4&#xB294; epitope &#xAE30;&#xBC18; &#xBC31;&#xC2E0;&#xACFC; &#xD30C;&#xC0C1;&#xD48D;&#xC5D0; &#xB300; &#xD55C; &#xCE58;&#xB8CC; &#xD56D; &#xCCB4;&#xC758; &#xAC1C;&#xBC1C;&#xC5D0; &#xB300; &#xD55C; &#xC88B;&#xC740; &#xD6C4;&#xBCF4;.

&#xD30C;&#xC0C1;&#xD48D; &#xB3C5; &#xC18C; &#xBCF4;&#xD638; &#xB2E8;&#xC77C; &#xD074;&#xB860; &#xD56D; &#xCCB4;&#xC5D0; &#xC758;&#xD574; &#xC778;&#xC2DD;&#xC5D0;&#xC11C; &#xC18C;&#xC124; &#xC120;&#xD615; epitope&#xC758; &#xC2DD;&#xBCC4;: &#xBC31;&#xC2E0; &#xB514;&#xC790;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xC758;&#xBBF8;.

T&eacute;tanos, una enfermedad infecciosa grave, es causado por la toxina tet&aacute;nica (TT) de Clostridium tetani, que sigue siendo uno de los problemas m&aacute;s cr&iacute;ticos de la salud sin resolverse a pesar de las estrategias preventivas. El terminal de carboxilo de TT (TTC) es responsable por la Uni&oacute;n de TT a las neuronas y su toxicidad y ha demostrado para ser inmunog&eacute;nica y protecci&oacute;n en diversas formas. Por lo tanto ser&iacute;a muy interesante identificar el epitopo en TTC a nivel molecular. En este estudio, hemos generado un anticuerpo de monoclonal neutralizante, 5 4, que inhibe la Uni&oacute;n de TT a su receptor y era eficaz protector 73,7 IU/mg. Por otra parte, 5 4 reconoce un epitopo lineal novela en TT, a saber, TC((1155-1171)), que se extiende desde Lys1155 a Val1171. Adem&aacute;s, TC((1155-1171)) fue demostrado para provocar la producci&oacute;n de un suero IgG que proteg&iacute;a a ratones contra un desaf&iacute;o con TT. Estos resultados sugirieron que TC((1155-1171)) y el anticuerpo monoclonal C 5 4 son buenos candidatos para el desarrollo de vacunas basadas en el epitopo y anticuerpos terap&eacute;uticos contra el t&eacute;tanos.

Identificaci&oacute;n de un epitopo novela lineal en toxina tet&aacute;nica reconocida por un anticuerpo monoclonal protectora: implicaciones para el dise&ntilde;o de la vacuna.

Infections due to Pseudomonas aeruginosa (PA) are becoming a serious threat to patients in intensive care units. A PA vaccine is a practical and economical solution to solve the problems caused by PA infection successfully. In recent years, several antigen candidates have been tested in animal and human clinical trials, but none of them has been approved to date. An alternative strategy for antigen screening and protective antigens is in urgent demand. In this study, we generated a genome-wide library of PA protein fragments tagged with maltose-binding protein (MBP). Using sera from patients who recovered after PA infection, we identified a novel protective antigen, FlgE, which is the structural component of the flagella hook. Vaccination with recombinant FlgE (reFlgE) induced a Th2-predominant immune response and reduced bacterial load and inflammation in PA-infected mice. Anti-reFlgE antibodies recognized native FlgE on the bacterial membrane in vitro and conferred protection in mice, which may be due to the mediation of opsonophagocytic killing and inhibition of bacterial motility. In addition, the combination of reFlgE with rePcrV, an engineered antigen we reported previously, provided elevated protection against PA infection. Our data demonstrate that FlgE is a promising vaccine candidate for PA and provide a new strategy for the efficient screening of antigens of other pathogens.

Flagella hook protein FlgE is a novel vaccine candidate of Pseudomonas aeruginosa identified by a genomic approach.

Epitope peptides are not suitable for nasal administration immunity due to their poor immunogenicity and low delivery efficiency. Here, we reported an intranasal self-assembled nanovaccine (I-OVA NE), which was loaded with the peptides IKVAV-OVA (I-OVA), a laminin peptide (Ile-Lys-Val-ala-Val, IKVAV) and OVA epitope conjugated peptide. This nanovaccine with I-OVA at a concentration of 4 mg/mL showed the average particle size of 30.37 ± 2.49 nm, zeta potential of -16.67 ± 1.76 mV, and encapsulation rate of 84.07 ± 7.59%. Moreover, the mucin did not alter its stability (size, PdI and zeta potential). And it also had no obvious acute pathological changes neither in the nasal mucosa nor lung tissues after nasal administration. Meanwhile, the antigen uptake of I-OVA NE was promoted, and the nasal residence time was also prolonged in vivo. Besides, the uptake rate of this nanovaccine was obviously higher than that of free I-OVA (P < 0.001) after blocking by the integrin antibody, suggesting that the binding of IKVAV to integrin is involved in the epitope peptide uptake. Importantly, this nanovaccine enhanced peptide-specific CD8T cells exhibiting OVA-specific CTL activity and Th1 immune response, leading to the induction of the protective immunity in E.G7-OVA tumor-bearing mice. Overall, these data indicate that I-OVA NE can be an applicable strategy of tumor vaccine development.

A novel self-assembled epitope peptide nanoemulsion vaccine targeting nasal mucosal epithelial cell for reinvigorating CD8 T cell immune activity and inhibiting tumor progression.

Vaccination strategies for rapid protection against multidrug-resistant bacterial infection are very important, especially for hospitalized patients who have high risk of exposure to these bacteria. However, few such vaccination strategies exist due to a shortage of knowledge supporting their rapid effect. Here, we demonstrated that a single intranasal immunization of inactivated whole cell of  elicits rapid protection against broad -infected pneumonia via training of innate immune response in  mice. Immunization-trained alveolar macrophages (AMs) showed enhanced TNF-α production upon restimulation. Adoptive transfer of immunization-trained AMs into naive mice mediated rapid protection against infection. Elevated TLR4 expression on vaccination-trained AMs contributed to rapid protection. Moreover, immunization-induced rapid protection was also seen in  and  pneumonia models, but not in  and  model. Our data reveal that a single intranasal immunization induces rapid and efficient protection against certain Gram-negative bacterial pneumonia via training AMs response, which highlights the importance and the possibility of harnessing trained immunity of AMs to design rapid-effecting vaccine.

Weijun Zhang

National Engineering Research Center of Immunological Products,
Department of Microbiology and Biochemical Pharmacy,
National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy

Third Military Medical University

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles