A Novel Design of Multi-epitope Vaccine Against Helicobacter pylori by Immunoinformatics Approach

International Journal of Peptide Research and Therapeutics - Helicobacter pylori (H. pylori) is a gram-negative spiral bacterium that caused infections in half of the world’s population and...     

幽门螺杆菌HspA融合蛋白口服疫苗的构建

构建表达幽门螺杆菌的保护性抗原分热休克蛋白A亚单位(HspA）和霍乱毒素B亚单位（CtxB）的重组融合蛋白的生物工程菌株,以此制备幽门螺杆菌的口服疫苗。用PCR方法扩增hspA和ctxB两个目的的基因片段,将它们分别克隆至pSK（＋）质粒上,然后插入含T7启动子ET－22b（＋）的表达载体中,构建嗓基因的表达质量pET－hct,转化E.coliBL21(DE3）,经IPTG诱导表达融合蛋白HCT。经测序,hspA-ctxB(hct)融合基因片段由726bp组成,可以编码242个氨基酸残基的多肽。经SDS－PAGE和免疫印迹分析检测发现,融合基因表达的蛋白质相对分子质量约为30kD。融合蛋白经镍离子柱纯化、复性后,和HspA共同标记同位素^125I,然后给小鼠灌胃,结果观察到HCT组小鼠血清中的^125I的放射量要明显高于HspA组（P＜0．001）,且吸收峰值时间明显提前。融合蛋白中的CtxB可明显促进小鼠对HspA的吸收,HCT融合蛋白可以作为预防和治疗幽门螺杆菌感染的侯选口服疫苗。     

The Molecular Characterization and Immunity Identification of Rhoptry Protein 22 of Toxoplasma gondii as a DNA Vaccine Candidate Against Toxoplasmosis

Toxoplasma gondii (T. gondii) rhoptry proteins (TgROPs) have been considered main targets and indicator molecules for immune diagnosis and prophylaxis since they initially present during the process of invasion. In this study, the effect of intramuscularly injecting the genetic vaccine pVAX‐ROP22 was evaluated, made by inserting the TgROP22 sequence into the eukaryotic expression vector of pVAX I, into BALB/c mice. The levels of IgG, IgG1, and IgG2a in pVAX‐ROP22 vaccinated animals were integrally increased. It was uncovered by cytokine profile analyses that the levels of IFN‐γ and IL‐2 were significantly increased, while no significant changes were detected in IL‐4 and IL‐10 levels. In addition, we found that immunization with pVAX‐ROP22 significantly prolonged the survival time (13.80 ± 1.75 d) of mice after challenge infection with the virulent T. gondii RH strain, in comparison with those of control animals (died within 10 d). Moreover, the number of brain cysts (1,406 ± 277) in the animals subjected to pVAX‐TgROP22 vaccination decreased remarkably (P < 0.05) compared with the blank control mice (2,333 ± 473), and the size of brain cysts in pVAX‐TgROP22 group was significantly smaller than the groups of blank, PBS and pVAXI. These results suggested that TgROP22 as DNA vaccine could trigger strong humoral and cellular responses and induce partial protection against toxoplasmosis.     

幽门螺杆菌尿素酶B单克隆抗体的制备与鉴定

目的:制备稳定分泌抗幽门螺杆菌尿素酶B单克隆抗体(mAb)的杂交瘤细胞系,并对其分泌的mAb进行鉴定。方法:用初步纯化的重组幽门螺杆菌尿素酶B免疫BALB/c小鼠,利用杂交瘤技术制备抗尿素酶B的mAb,用间接ELISA检测mAb的特异性和亲和力,检测mAb腹水效价,鉴定Ig亚类并测定其抗原决定簇。结果:获得8株能稳定分泌抗尿素酶B的mAb杂交瘤细胞系,这8株单抗与能产生尿素酶的小肠结肠耶尔森氏菌、肺炎克雷伯氏菌和普通变形杆菌均无交叉反应,相对亲和力为1.13×10-8～4.66×10-10,腹水mAb效价可达2×104～3.2×105。其中2株单抗属IgG1亚类,3株单抗属IgG2a亚类。8株单抗分属于3种不同的抗原决定簇。结论:获得了IgG1和IgG2a类型的针对3种不同抗原决定簇的特异性幽门螺杆菌尿素酶B的mAb,为进一步用于幽门螺杆菌的临床诊断和实验研究创造了条件。     

Subtractive Proteomics and Immuno-informatics Approaches for Multi-peptide Vaccine Prediction Against Klebsiella oxytoca and Validation Through In Silico Expression

International Journal of Peptide Research and Therapeutics - Klebsiella oxytoca is a&nbsp;gram-negative bacterium. It is opportunistic in nature and causes hospital acquired infections....     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.     

幽门螺杆菌热休克蛋白A基因的克隆、表达及免疫原性研究

幽门螺杆菌的感染可诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ（ＨｓｐＡ）可刺激机体产生保护性的免疫反应。用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序ＨｓｐＡ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。ＳＤＳＰＡＧＥ和免疫印迹分析检测发现,ＨｓｐＡ基因表达的蛋白质分子量约为１５ｋＤ,并证实该重组蛋白质可以被幽门螺杆菌感染阳性患者的血清所识别,同时将其免疫小鼠可刺激机体产生抗该重组蛋白质的抗体。ＨｓｐＡ有可能作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗。     

幽门螺杆菌vacA基因的克隆和序列分析

空泡毒素是幽门螺杆菌产生的已知的唯一蛋白毒素,该毒素与感染者胃肠上皮务和溃疡形成密切相关,同时也是幽门螺杆菌免疫预防和免疫治疗的重要候选组分。从幽门螺杆菌NCTC11637染色体DNA中经PCR方法获得了2.9kb的该基因成熟肽全长序列,将该基因克隆至载体pET22b ,经PCR扩增和酶切鉴定后序列分析表明,该基因与已知序列完全一致。     

Helicobacter pylori Identification: A Diagnostic/Confirmatory Method for Evaluation

The Helicobacter pylori extra gastric reservoir is probably the oral cavity. In order to evaluate the presence of this bacterium in patients with periodontitis and suspicious microbial cultures, saliva was collected from these and non-periodontitis subjects. PCRs targeting 16S rRNA gene and a 860 bp specific region were performed, and digested with the restriction enzyme DdeI. We observed that the PCR–RFLP approach augments the accuracy from 26.2 % (16/61), found in the PCR-based results, to 42.6 % (26/61), which is an excellent indicator for the establishment of this low-cost procedure as a diagnostic/confirmatory method for H. pylori evaluation.     

幽门螺杆菌AlpA基因中四种黏附素基因保守区的克隆、表达、纯化及鉴定

幽门螺杆菌（Helicobacter pylori,Hp）感染是慢性活动性胃炎和消化性溃疡的主要病因,与胃腺癌、胃黏膜相关淋巴样组织（MALT）淋巴瘤的发生亦密切相关.鉴于Hp已证实的四种粘附素保守区（AB）是外膜蛋白（OMP）和膜孔素(porin)样成分,而外膜蛋白和膜孔素样成分是优秀的疫苗候选抗原.用PCR技术扩增AB基因,将其定向插入pET-22b（+）载体,在BL21（DE3）大肠杆菌中表达.测序显示AB基因长588 bp,编码195个氨基酸.SDS-聚丙烯酰胺凝胶电泳和凝胶扫描分析,AB基因表达的蛋白质分子质量约为22.5 ku,其重组蛋白质表达量占菌体总蛋白质的29%,表达产物经亲和层析纯化后蛋白质纯度达96%.经免疫印迹证实该重组蛋白可以被AlpA免疫兔血清所识别.AB蛋白的获得为进一步研究Hp黏附素保守区的分子黏附机制和免疫防治作用提供了基础.     

Cloning, Expression, and Enzymatic Characterization of Isocitrate Dehydrogenase from Helicobacter pylori

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k _cat of 87 s^?1, K _m of 124 μM and k _cat/K _m of 7 × 10⁵ M^?1s^?1 toward isocitrate, k _cat of 80 s^?1, K _m of 176 μM and k _cat/K _m of 4.5 × 10⁵ M^?1s^?1 toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 °C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection.     

HPV16-E7 Protein T Cell Epitope Prediction and Global Therapeutic Peptide Vaccine Design Based on Human Leukocyte Antigen Frequency: An In-Silico Study

International Journal of Peptide Research and Therapeutics - Cervical cancer is the second most common leading cause of women's death due to cancer worldwide, about 528,000 patients’...     

人参皂苷生物合成相关新基因GBR6的cDNA克隆及序列分析

从人参根组织中分离总RNA,使用RT—PCR扩增出人参皂苷生物合成相关基因GBR6开放阅读框(ORF)cDNA片断,并将其定向克隆入原核高效表达载体pQE30,阳性克隆经BamHⅠ和PstⅠ双酶切鉴定、测序验证与已知的GBR6ORF序列完全一致．因而成功构建了pQE30-GBR60RF融合原核表达载体,为下一步进行GBR6功能表达分析奠定了基础．     

一个新鼻咽癌抑瘤候选基因的克隆及其功能初步分析

从cDNA 代表差异分析法（cDNA representational difference analysis, cDNA RDA）分离的新cDNA片段入手,进一步采用RT-PCR验证,其中发现AF152605片段在74%鼻咽癌(nasopharyngeal carcinoma,NPC)活检组织中表达下调和缺失,DNA印迹显示其代表一转录本为2.1 kb的基因,结合生物信息学,运用文库筛选克隆该基因,命名为NAG4基因,GenBank登录号AF179285,定位于6q22.1～22.33,至少含有两个外显子,并在第一外显子的上游有TATA盒样序列,编码一个508个氨基酸组成的、分子质量为57.4 ku的蛋白质;功能预测NAG4基因产物与小鼠溴区蛋白BP75有84%同源,是含有多个磷酸化位点和溴区结构域的核内转录因子;突变分析表明NAG4基因在HeLa细胞株中发生整码突变.以上结果说明NAG4基因是鼻咽癌抑瘤基因的良好候选者,其表达的下调参与了鼻咽癌的发生.     

The Synthetic Plasmodium falciparum Circumsporozoite Peptide PfCS102 as a Malaria Vaccine Candidate: A Randomized Controlled Phase I Trial

Background

Fully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102) in malaria naive adults.

Methodology and Principal Findings

Thirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 µg) and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity) was based on the frequency of adverse events (AE) and of abnormal biological safety tests; secondary-end point (immunogenicity) on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema). After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-γ production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-γ secreting CD8⁺ T cell responses. Responses were only marginally boosted after the 3^rd vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 µg was less immunogenic in comparison to 30 and 100 µg that induced similar responses. AS02A formulations with 30 µg or 100 µg PfCS102 induced about 10-folds higher antibody and IFN-γ responses than Montanide formulations.

Conclusions/Significance

PfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential for protection. Two or three immunizations with a dose of 30 µg formulated with AS02A appeared the most appropriate choice for such studies.

Trial Registration

Swissmedic.ch 2002 DR 1227     

A Novel Mucosal Vaccine Against Foot-and-Mouth Disease Virus Induces Protection in Mice and Swine

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6×His-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1–hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1–hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10⁵ FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1–hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10^6.5 FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV. Houhui Song, Zhiliang Wang and Dongxia Zheng contributed equally to this work.     

Peptide Folding Problem: A Molecular Dynamics Study on Polyalanines Using Different Force Fields

In the last decade many advances have been made on molecular dynamics simulations and different force fields were developed from the combination of differentiable functions of the atomic coordinates to represent the system energy and of parameters that describe the geometric and energetic properties of inter-particle interactions. However, it has been shown that very subtle modifications to commonly used molecular mechanical potentials can significantly alter the behavior of those potentials inducing stabilizing or destabilizing effects in the patterns of peptides or proteins. In this article we describe the behavior of polyalanine peptides under the influence of various “force fields”. The polyalanines were chosen as study model since their structural features were already studied experimentally and thus our computational results were easily comparable with the experimental ones. In particular, three peptides composed of 8, 10 and 12 alanine residues were subjected to molecular dynamics simulations using 12 different force fields to understand what is the most appropriate force field to properly simulate their folding. Our results showed that Amber99? is the best force field able to generate helical conformations in agreement with experimental data.