Faris Robert, Weber Mary M
Microbiology and Immunology, University of Iowa, Carver College of Medicine, Iowa City, U.S.A.
Bio Protoc. 2019 Dec 20;9(24):e3459. doi: 10.21769/BioProtoc.3459.
) is an obligate intracellular pathogen that cannot be cultured axenically and must be propagated within eukaryotic host cells. There are at least 15 distinct chlamydial serovariants that belong to 2 major biovars commonly referred to as trachoma and lymphogranuloma venereum (LGV). The invasive chlamydia LGV serovar L2 is the most widely used experimental model for studying biology and infection and is the only strain with reliable genetic tools available. New techniques to genetically manipulate L2 have provided opportunities to make mutants using TargeTron and allelic exchange as well as strains overexpressing epitope-tagged proteins, in turn necessitating the regular purification of transformant and mutant clones. Purification of is a labor-intensive exercise and one of the most common reagents classically used in the purification process, Renografin, is no longer commercially available. A similar formulation of diatrizoate meglumine called Gastrografin is readily available and we as well as others have had great success using this in place of Renografin for chlamydial purifications. Here, we provide a detailed general protocol for infection, propagation, purification, and titering of serovar L2 with additional notes specifically pertaining to mutants or recombinant DNA carrying clones.
（衣原体）是一种专性细胞内病原体，无法在无细胞环境中培养，必须在真核宿主细胞内繁殖。至少有15种不同的衣原体血清变种，它们属于通常被称为沙眼和性病性淋巴肉芽肿（LGV）的2个主要生物变种。侵袭性衣原体LGV血清变种L2是研究生物学和感染最广泛使用的实验模型，并且是唯一拥有可用可靠遗传工具的菌株。对L2进行基因操作的新技术提供了使用TargeTron和等位基因交换制造突变体以及过表达表位标记蛋白的菌株的机会，这反过来又需要定期纯化转化体和突变体克隆。衣原体的纯化是一项劳动密集型工作，纯化过程中经典使用的最常见试剂之一泛影葡胺已不再商业化供应。一种名为 Gastrografin 的类似泛影葡胺制剂很容易获得，我们以及其他人使用它替代泛影葡胺进行衣原体纯化取得了巨大成功。在这里，我们提供了一份关于血清变种L2感染、繁殖、纯化和滴定的详细通用方案，并附带了专门针对携带突变体或重组DNA的克隆的注释。
