百日咳毒素在疫苗中的研究现状及应用

百日咳毒素(PT)是百日咳杆菌所产生的主要毒力因子,具有多种生物学活性,是无细胞百日咳疫苗中唯一不受争议的抗原成分.随着分子生物学技术的发展,研究者试图开发与PT有关的重组亚单位疫苗、重组细菌疫苗和DNA疫苗.本文对近年来的研究进展作一综述.     

超抗原VEGF-SEA融合基因的克隆与表达

目的:构建血管内皮生长因子-葡萄球菌肠毒素A(VEGF-SEA)基因的原核表达质粒,获得VEGF-SEA融合蛋白。方法:制备VEGF-SEA基因片段,插入pET40b构建重组质粒pET40b-VEGF-SEA,经序列分析正确后转化大肠杆菌BL21(DE3),进行IPTG诱导表达蛋白。用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达蛋白的相对分子质量及表达形式,并对产物采用His·Bind Buffer kit试剂盒纯化。结果:获得VEGF-SEA基因片段长1130bp,经序列分析与预期完全一致,其表达出目的蛋白的大小约为66.2ku,以包涵体形式表达,经纯化,纯度达到90%以上。结论:表达载体pET40b-VEGF-SEA构建成功,VEGF-SEA融合蛋白获得高效表达,本实验为进一步研究VEGF-SEA蛋白的活性及其功能,探讨超抗原抑制肿瘤生长作用奠定了基础。     

可溶性组织因子的基因克隆、表达与性质研究

组织因子是外源性凝血途径的启动因子，为膜受体蛋白，胞外区的促凝血功能几乎与完整分子相同，因此采用重组DNA技术制备组织因子的胞外区分子，并对其活性进行研究，以便替代兔脑粉用于PT检测。以人胎盘总RNA为模板，用RT－PCR扩增组织因子胞外区cDNA基因，经序列分析证实后大肠杆菌中表达，产物形成包涵体。表达产物经复性，离子交换和凝胶过滤色谱纯化后与磷脂结合，进行促凝血试验。结果表明在磷脂存在下，可溶性TF有明显的促凝血活性，可以取代兔脑粉有于PT检测。     

Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes

The adenylate cyclase toxin, CyaA, is one of the key virulent factors produced by Bordetella pertussis, the causative agent of whooping cough. This toxin primarily targets innate immunity to facilitate bacterial colonization of the respiratory tract. CyaA exhibits several remarkable characteristics that have been exploited for various applications in vaccinology and other biotechnological purposes. CyaA has been engineered as a potent vaccine vehicle to deliver antigens into antigen-presenting cells, while the adenylate cyclase catalytic domain has been used to design a robust genetic assay for monitoring protein–protein interactions in bacteria. These two biotechnological applications are briefly summarized in this chapter.     

大肠杆菌caiAB基因的克隆与表达

从E.coli MC4100菌株的染色体DNA中利用鸟枪法克隆含编码豆甜菜碱还原酶和L－肉碱脱水酶的caiAB基因片段，并经序列分析验证。由重组质粒pZJD-1480亚克隆得到pT7-A以及pT7-B重组表达质粒，后者转入E.coli BL21(DE3)菌株中，经异丙基硫代半乳糖苷（IPTG）诱导，在聚丙烯酰胺凝胶电泳（SDSPAGE）上分子质量为40ku附近可见明显的表达蛋白带。     

Physicochemical Characterization of Sabin Inactivated Poliovirus Vaccine for Process Development

Upscaling the production capacity of inactivated poliovirus vaccines (IPV) is urgently needed to eradicate polio worldwide. For the development of a robust manufacturing process for IPV, the impact of stresses on the properties of the poliovirus during manufacturing needs to be carefully evaluated. In this study, the physicochemical properties of Sabin poliovirus after low pH exposure were analyzed by asymmetrical flow field-flow fractionation coupled to multi-angle laser light scattering (AF4-MALS), sedimentation velocity analytical ultracentrifugation (SV-AUC), transmission electron microscopy (TEM), dynamic light scattering (DLS) and surface plasmon resonance (SPR). Low pH stress caused structural changes and aggregation of inactivated poliovirus virions, whereas degraded virion particles would not revert to native virions even after neutralization. Importantly, a complete loss of the D-antigenicity of IPV by low pH stress, followed by neutralization, was observed in SPR. These results suggest that the exposure of poliovirus particle to low pH stress would induce irreversible denaturation and aggregation of virus particles and lead to the loss of D-antigenicity; thus, low pH stress during the manufacturing of poliovirus vaccine should be minimized. The analytical methods above can be efficiently utilized in the development of high-integrity manufacturing processes and high-quality vaccines.     

Kininogen D5和TRAIL融合蛋白的克隆表达及生物学活性的研究

用重组PCR技术得到Kininogen D5和TRAIL的融合编码序列,将该DNA片段克隆到原核表达载体pMAL-c2,重组质粒转化大肠杆菌BL21,IPTG诱导蛋白表达,得到MBP-KT和MBP-TK,经AmyloseResin亲和层析柱层析,得到初步纯化的融合蛋白.结果表明MBP-KT对胰腺癌细胞1990有明显的杀伤作用其作用的ED5.为20 ng/ml,而MBP-TK对1990的抑制作用不明显;同时,MBP-KT和MBP-KD5对内皮细胞ECV304有明显的抑制作用,MBP/KT作用于ECV304的ED50为0.1μg/ml,MBP/KD5作用于ECV304的ED50为9.5μg/ml,但是MBP-TK和TRAIL几乎无作用,表明融合蛋白KT既具抗肿瘤作用又有抗新生血管的作用.本研究为进一步开发靶向性杀伤肿瘤药物奠定了基础.     

The Preparation,Characterization, and Evaluation of Cationic Microparticles for DNA Vaccine Delivery

Pharmaceutical Research -     

Physical Characterization and Formulation Development of a Recombinant Pneumolysoid Protein-Based Pneumococcal Vaccine

Streptococcus pneumoniae is a major cause of death in children worldwide. There are more than 90 known pneumococcus serotypes that vary by geographical location. Pneumolysin is a protein toxin produced by virtually all invasive strains of S. pneumoniae and is considered an important virulence factor. Pneumolysin is immunogenic and has the potential to be a new vaccine antigen offering broad serotype-independent coverage. To develop a stable vaccine formulation, the conformational stability of a recombinant pneumolysin mutant (pneumolysoid L460D) was characterized by various techniques. Three data visualization diagrams were constructed to summarize the biophysical data of the L460D pneumolysoid; the protein is most stable in solution at pH 6–7, and loses conformational integrity above 48°C. Excipient screening assays were performed and sugars such as trehalose and sucrose stabilized the pneumolysin mutant with respect to improving thermal transition temperatures and minimizing aggregation. In addition, the protein antigen showed efficient binding to aluminum hydroxide adjuvant. The conformational stability of the L460D pneumolysoid on the surface of alhydrogel adjuvant was little affected by adsorption, either with or without excipients. These studies provide important preformulation characterization information useful for the development of a stable pneumolysin mutant-based vaccine.     

Characterization of Glutamate Receptor by Spider Toxin

Abstract

Recent study has shown that the venom of some orb-web spiders contain potent blockers of the glutamate receptors. Joro spider toxin (JSTX) derived from Nephila clavata has been found to block excitatory postsynaptic potentials and glutamate-evoked responses in the neuromuscular synapse of crustacea, the squid giant synapse and the mammalian brain synapse. Structures of the toxins (JSTXs, NSTXs) of spiders belonging to the genus Nephila were determined and it was found that a unique 2,4-dihydroxyphenylacetyl asparaginyl cadaverine part was conserved between all toxins, indicating that this part is intimately involved in the blocking activity.

Labeling of synthesized JSTX-3 was used for histological investigation of glutamate receptors. Using autoradiography ¹²⁵I-JSTX-3 was found to bind at the lobster neuromuscular synapse. A histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat hippocampus and cerebellum. JSTX-3 was used for isolation of glutamate receptors from brain. A crude synaptic membrane fraction from rat hippocampus and cerebellum was solubilized by Triton X-100. SDS-PAGE of the affinity purified JSTX-3 binding proteins showed at least 4 bands around 70 K daltons.     

Structural Characterization and Formulation Development of a Trivalent Equine Encephalitis Virus-Like Particle Vaccine Candidate

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ～70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ～50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from ?80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at ?20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.     

A Rational, Systematic Approach for the Development of Vaccine Formulations

With the continuous emergence of new infectious diseases and new strains of current diseases, such as the novel H1N1 influenza in 2009, in combination with expanding competition in the vaccine marketplace, the pressure to develop vaccine formulations right the first time is increasing. As vaccines are complex, costly, and have high risk associated with their development, it is necessary to maximize the potential for development of a successful formulation quickly. To accomplish this goal, the historical empirical approach to formulation development needs to be updated with a rational, systematic approach allowing for more rapid development of safe, efficacious, and stable vaccine formulations. The main components to this approach are biophysical characterization of the antigen, evaluation of stabilizers, investigation of antigen interactions with adjuvants, evaluation of product contact materials, and monitoring stability both in real time and under accelerated conditions. An overview of investigations performed for each of these components of formulation development is discussed. The information gained in these studies is valuable in forming the base of knowledge for the design of a robust formulation. With the use of continually advancing technology in combination with maintaining a rational, systematic approach to formulation development, there is a great increase in the probability of successfully developing a safe, effective, and stable vaccine formulation.     

Vaccinomics: Current Findings, Challenges and Novel Approaches for Vaccine Development

Recent years have witnessed a growing interest in a field of vaccinology that we have named vaccinomics. The overall idea behind vaccinomics is to identify genetic and other mechanisms and pathways that determine immune responses, and thereby provide new candidate vaccine approaches. Considerable data show that host genetic polymorphisms act as important determinants of innate and adaptive immunity to vaccines. This review highlights examples of the role of immunogenetics and immunogenomics in understanding immune responses to vaccination, which are highly variable across the population. The influence of HLA genes, non-HLA, and innate genes in inter-individual variations in immune responses to viral vaccines are examined using population-based gene/SNP association studies. The ability to understand relationships between immune response gene variants and vaccine-specific immunity may assist in designing new vaccines. At the same time, application of state-of-the-art next-generation sequencing technology (and bioinformatics) is desired to provide new genetic information and its relationship to the immune response.     

胰高血糖素基因的合成、克隆与融合表达

选用Ｅ．ｃｏｌｉ偏爱的密码子，用计算机辅助设计合成了四段寡核苷酸序列，通过二步ＰＣＲ法构建胰高血糖皮素基因，总长１２４ｂｐ，并直接克隆在ｐＵＣ１８质粒中，序列分析证实合成与克隆的胰高血糖素基因序列与设计完全相符，将该基因引入质粒ｐＫＡ，使胰高糖素在ＡｎｓＢ的Ｃ端，实现融合表达，用ＥＬＩＳＡ和ＳＰＲ分别检测表达产物。     

条纹斑竹鲨鱼DNA结合抑制因子cDNA的克隆、序列特征及其在肝再生过程中的表达分析

DNA结合抑制因子(Inhibitor of DNA binding,Id)又称分化抑制因子(Inhibitors of Differentiation,Id),属于负调控因子HLH家族的成员,其功能是与DNA结合并抑制细胞的分化和促进细胞的增殖。为揭示Id蛋白在鲨鱼肝脏再生过程中的作用,作者从前期构建的条纹斑竹鲨鱼肝脏cDNA文库中克隆到编码Id蛋白的cDNA全序列并预测了其编码产物(CpId)的结构,探讨了该基因在鲨鱼体内的时空表达方式,结果表明:CpId cDNA全长为1 074 bp,编码一个由135个氨基酸残基组成的无跨膜区的胞内蛋白,理论分子质量(Mr)为14 857.2,理论等电点(pI)为5.35。CpId蛋白序列与其他生物来源的Id蛋白的同源性介于42%～62%之间,其N端第31～87 aa组成一个相对保守的Myc型helix-loop-helix结构域。CpId基因在鲨鱼各种组织中均有表达,但以在肝脏、脑和生殖腺中的表达最高;肝部分切除后,CpId基因的表达迅速上调,在切除1 h后到达最高值(为正常肝组织的18倍左右),其后逐渐下降,并在24 h时恢复至正常肝组织中的水平;CpId基因的表达在肝部分切除后的第48 h出现第2个相对较高的表达峰。上述结果表明CpId基因参与了肝脏再生调控的过程,其作用可能与促进肝细胞的增殖再生过程有关。     

我国疫苗生产用动物和动物源性材料安全标准现状的分析

目的 分析我国疫苗生产用动物和动物源性材料质量要求与国外相关技术规范的差异,探索进一步提高和完善我国疫苗生产用动物和动物源性材料质量标准的途径.方法 概述我国实验动物质量标准和局限性,将《中国药典》与国外相关技术法规对疫苗生产用动物和动物源性材料质量标准进行对比分析.结果与结论 我国应研究制订达到或接近国际标准要求的疫苗生产用动物源性材料质量标准,保证疫苗质量和公众用药安全.     

新型降钙素的基因合成与克隆表达

借助计算机辅助设计了一种新的人降钙素类似物（新型降钙素）。根据密码子偏爱性原理及基因操作原则了它的基因,运用化学法和酶法相结合的技术合成了该基因,并克隆到质粒ｐＵＣ１９中,ＤＮＡ测序验证正确。该基因被克隆到融合型表达本ｐＧＥＸ－２Ｔ中,重组融合型表达载体ｎＣＴ／ｐＧＥＸ－２Ｔ转化Ｅ．ｃｏｌｉＢＬ２１,增减表达,ＳＤＳ－ＰＡＧＥ分析可见Ｍｒ约３１０００的融合蛋白带,融合蛋白占细胞内总蛋白３４％。Ｗｅ     

RGD修饰多肽EDSM-Y的克隆、表达、纯化和初步活性研究

固相合成多肽EDSM-Y在体内外具有良好的抗血管生成和抗肿瘤的活性,该研究希望通过基因工程重组手段获得目的蛋白EDSM-Y,为进一步研究开发奠定基础。PCR扩增得到目的基因,连接入原核表达载体;以IPTG诱导实现外源基因表达,SDS-PAGE和Western Blot验证;摸索发酵表达纯化条件,分离目的蛋白;细胞增殖实验检测EDSM-Y的活性。成功构建了pET-23a(+)-EDSM-Y重组表达载体,但目的蛋白表达量较低。后将EDSM-Y克隆入pET-32a(+)重组表达载体进行融合表达,改变表达载体后目的蛋白表达量显著增加。目的蛋白经过先后两次亲和层析,纯度分别可到达60%和85%,活性实验表明EDSM-Y对血管内皮细胞具有增殖抑制作用。通过基因工程重组能获得具有抑制细胞增殖活性的抗肿瘤多肽EDSM-Y。