Name: forward genetic approaches in chlamydia trachomatis
Uploaded: 2013-10-23T18:30:00
Description: Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

1。化学诱变注：我们发现该复制的RB的形式是比EB形式更适合于化学诱变。在周期中（18至20 HPI），苏格兰皇家银行是人数最多的前RB-EB过渡。由于沙眼衣原体是一种专性细胞内病原体，宿主健康的影响诱变可以限制细菌复苏。 Vero细胞被发现是有抵抗性的高层次的EMS比其他测试的细胞系的不利影响。 通过重组突变分离需要选择对抗生素有抗药性的重...

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这种方法符合遗传分析的基本要求，因为它建立了基因型和表现型之间的联动。重要的是，这是实现不借助常规的分子工具的重组DNA转化的插入失活的基因在细菌中，这通常是在非模型的微生物的基因的功能分析的限速步骤。 一个关键的步骤是，以确保克隆的噬菌斑纯化的突变体。与野生型或突变“钳工”的交叉污染，可以迅速导致突变株被outcompeted。同样，测绘非克隆样本...

有一个独特的专性细胞内细菌沙眼衣原体占估计有280万生殖道感染，每年在美国（疾病控制中心），相关的后遗症，如盆腔炎，异位妊娠，不孕不育（1） 衣原体属生理发育周期的两相组成的两种形式：感染性但非复制生小体（EB）和非感染性的，但复制的网状体（RB）。感染开​​始，随后者endoctyotosis（2）的上皮细胞的胚体附件。称为膜结合液泡内夹杂物，EBS分化成RB形式，然后复制二进制裂变的。时周期中，资源块的转换回成胚体，然后将其排出到细胞外空间，开始新一轮的感染的宿主细胞裂解（3）。 沙眼衣原体是难治性标准的分子遗传工具，如针对性的基因置换，转座子，转导噬菌体，大多数细菌遗传学的研究中心一直与常规操作，目前还不清楚在何种程度上个别衣原体基因有助于先天免疫逃避的，营养的收购，发育的过渡，或其他进程在真核宿主（4）重要的病原体的生存。因此，这种病原体仍然很差的特点，尽管其临床重要性。 衣原体菌的基因组。都比较小（〜1 MB）（5）采用新一代测序技术测序的多个物种和生物型。比较基因组分析由WGS衣原体种的演变，并使其适应人类（6-8），并在一定程度上提供了独特的见解，提供了一些有关的潜在的毒力因子的功能（9，10）。 Ť他显示临床分离株的遗传多样性并不提供系统最毒力因子的功能映射所需的分辨率，这大概是因为在这样的基因的突变会被容易地选择反对。如果没有从自然选择，诱变诱发的基因变异，加上有明确的检测措施缺陷致病，可扩展的突变谱，可以调查的混杂影响。化学诱变剂，尤其是有用的，因为它们可以产生空的，有条件的，减少亚效等位基因（功能），和hypermorphic（增益函数）等位基因。随着功能强大的下一代基因组测序技术的到来，这种突变可以很容易地识别和映射。以这种方式，可以强关联之间的突变的基因或遗传途径和一个共同的表型，使正向遗传学的方法中的应用。 临床分离株的基因组序列的嵌合透露between血清型和频繁的重组位点（11）。重组的实证证据证明，通过共同感染两种不同的抗生素有抗药性的菌株和选择双抗重组的后代，这是显露有两种菌株的遗传贡献（12，13）。因此，在一个共同感染的设置的野生型和突变株之间的遗传交换允许针对受影响的偏析化学诱导突变导致所观察到的表型的基因，该基因。 这里，我们描述了一种方法来执行衣原体基因分析在化学诱变，WGS感染细胞内的DNA交换（14）（ 图1），以及系统的基础上的。

<table border="0" cellspacing="0" cols="4">
	<colgroup width="291">
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	<colgroup width="123">
	</colgroup>
	<colgroup width="144">
	</colgroup>
	<colgroup width="84">
	</colgroup>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate (EMS)</td>
			<td>Sigma</td>
			<td>M0880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclohexamide</td>
			<td>Sigma</td>
			<td>C4859-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) solution with 0.493 mM MgCl2 and 0.901 mM CaCl2 (PBS+MgCl2/CaCl2)</td>
			<td>Life Technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5x SPG buffer (1.25 M sucrose, 50 mM sodium phosphate, 25 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water (sterile, tissue culture grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x DMEM (prepared from powder, buffered with 7.4 g/L Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>D7777</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids (NEAA)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2% GTG Agarose , autoclaved</td>
			<td>Lonzo</td>
			<td>50070</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA purification kits</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Life Technologies</td>
			<td>10503-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (molecular biology grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 mM NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 M HEPES ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 tissue culture flasks (T25 flasks)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Centrifuge and adaptors for spinning tissue plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Dissection microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer (Qubit)</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>Adaptive Focused Acoustics S220 instrument</td>
			<td>Covaris</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

forward genetic approaches in chlamydia trachomatis

沙眼衣原体 ，性病和眼部感染的病原体，仍然差的特点，由于其难解实验转型与重组DNA。我们开发了一种方法来进行遗传分析，C.尽管缺乏分子遗传工具衣原体 。我们的方法包括：）。化学诱变快速生成全面定义基因的突变体库，具有不同表型，II。）全基因组测序（WGS）映射潜在的遗传病变，找到突变基因（S）之间的关联一个共同的表型; III）代重组菌株通过哺乳动物细胞突变体和野生型细菌感染的合作。因此，我们能够建立基因型和表现型之间的因果关系。耦合化学诱导的基因变异与WGS建立相关的基因型 - 表型关联寿LD是广泛适用于大名单，医疗和环境重要的微生物，目前棘手的遗传分析。

暴露于诱变剂会导致夹杂物出现没有细菌，大概是由于细菌细胞死亡。通常情况下，血清型LGV-L2将完全溶解感染后48小时内受感染的细胞，但治疗与诱变这个周期延长到90小时。大约10％的夹杂物有望复苏。在我们的实验中，用20毫克/毫升EMS感染的Vero细胞导致在回收感染性子代，与未处理的对照组相比，减少99％。诱变剂治疗也导致斑块的出现改变形态，包括小斑块（SPQ）（ 图3）。其...

Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

Forward Genetic Approaches in Chlamydia trachomatis

我们描述了一种方法来进行遗传分析化学诱变和全基因组测序的基础上衣原体 。此外，感染细胞内的DNA交换系统可用于遗传作图描述。此方法可广泛适用于微生物系统，缺乏改造系统和分子遗传工具。

正向遗传途径<em&gt;沙眼衣原体</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

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Forward Genetic Approaches in Chlamydia trachomatis

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

科研

免疫与感染

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学：生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...