| Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi
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Author:
Date:
2020-09-05
[Abstract] Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion or growth capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition activity (GIA) ...
[摘要] [摘要] 疟疾仍然是全球发病率和死亡率的主要原因。该疾病的临床症状源于宿主血液中疟原虫的生长和繁殖。因此，体外测定以确定药物，抗体和遗传扰动如何影响疟原虫寄生虫的生长速率对于开发新疗法和增进我们对寄生虫生物学的理解至关重要。由于两个恶性疟原虫和P. knowlesi 可以在培养物中维持与人体红细胞，抗疟疾药物和抑制性抗体靶向的侵袭能力的影响疟原虫寄生虫 是通过使用针对这两个物种乘法测定或生长抑制测定法常规地研究。该协议给出了详细的一步一步的过程来进行基于所述寄生虫乳酸脱氢酶的活性为基础的流式细胞仪乘法测定和生长抑制活性测定法测试性中和抗体的疟原虫knowlesi 适于人类红血细胞培养物中。虽然类似测定法是用于很好地建立的恶性疟原虫，P. knowlesi 被更密切相关的所有其他人类感染性物种（帕切科等人。，2018），因此可以用作替代用于测试药物和疫苗用于其它疟疾种类，例如如间日疟原虫，它是非洲以外疟疾最广泛的病因，但尚未在实验室条件下进行培养。
[背景 ] ...
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| Construction of FWPV Chimaeric MVA
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Author:
Date:
2015-01-05
[Abstract] Construction of chimaeric MVA is a useful tool with which to study gene function of related viruses. The protocol given here describes MVA chimaeras containing genes from Fowlpox virus (FWPV), although this can be applied to DNA derived from other organisms. There are a number of steps required to make the chimaeric MVA: 1) Purification of viral particles; 2) Extraction of DNA from purified viral particles; 3) Assembly of linear recombination templates; 4) Transfection of linear recombination templates; 5) Selection of chimaeric MVA.
Note: This procedure uses live virus, and should be conducted using Good Microbiological Practice, in accordance with international and national biocontainment requirements. This procedure also involves Genetic Modification of microorganisms, and ...
[摘要] 嵌合MVA的构建是研究相关病毒的基因功能的有用工具。 本文给出的方案描述了含有来自鸡痘病毒（FWPV）的基因的MVA嵌合体，尽管这可以应用于来自其他生物体的DNA。 制备嵌合MVA需要许多步骤：1）病毒颗粒的纯化; 2）从纯化的病毒颗粒中提取DNA; 3）装配线性重组模板; 4）线性重组模板的转染; 5）嵌合MVA的选择。
注意：此程序使用活病毒，并应根据国际和国家生物承诺要求使用良好微生物实践进行。 该程序还涉及微生物的遗传修饰，并且在开始前应获得适当的安全批准。
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| Preparation of Parasite Protein Extracts and Western Blot Analysis
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Author:
Date:
2014-06-05
[Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available to achieve this. If the protein is present within the parasite or is attached to a cellular structure of the iRBC cell, saponin can be used. This reagent lyses the membranes of infected and uninfected erythrocytes, the Maurer´s clefts (vesicular structures in the iRBC) and the parasitophorous vacuole membrane containing the parasite but leaves the parasite plasma membrane intact, providing a convenient procedure to isolate intact parasites ...
[摘要] 为了制备用于蛋白质印迹分析的恶性疟原虫血液阶段的蛋白质提取物，需要将感染的红细胞（iRBC）与构成寄生虫的主体的未感染的红细胞（uRBC）分离文化。根据感兴趣的寄生虫蛋白的定位，可以使用不同的方法来实现这一点。如果蛋白质存在于寄生虫内或附着于iRBC细胞的细胞结构，则可以使用皂苷。该试剂裂解感染的和未感染的红细胞的膜，Maurer's裂口（iRBC中的囊泡结构）和含有寄生虫的寄生虫液泡膜，但是使寄生虫质膜完整，提供了一种方便的方法来分离没有uRBC的完整寄生虫。然而，该方法的缺点是iRBC的宿主细胞胞质和寄生泡（PV）含量丢失。如果这必须避免，可以使用Percoll梯度将完整的iRBC与uRBC分离。然后可以使用Tetanolysin和皂苷的顺序处理来从寄生虫选择性释放iRBC细胞溶质和PV含量。这些选择性裂解方法也适合于确定目标蛋白质的亚细胞定位
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