Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes

Current Protocols in Microbiology Pub Date : 2020-05-07 DOI:10.1002/cpmc.101

Samuel S. Porter, Alison A. McBride

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引用次数: 7

Abstract

This protocol describes the production of human papillomavirus (HPV)–derived quasiviruses. Quasiviruses are infectious particles that are produced in 293TT packaging cells and contain a complete viral genome. We describe methods for infection of primary human keratinocytes with HPV quasiviruses, as well as assays to measure early viral DNA replication and transcription. Published 2020. U.S. Government.

Basic Protocol 1: Transfection, harvest, and isolation of HPV quasiviruses

Alternate Protocol 1: Packaging HPV DNA replicated in 293TT cells

Alternate Protocol 2: Production of higher-purity quasivirus using the “Ripcord” method

Support Protocol 1: Production of HPV minicircles

Support Protocol 2: Production of recircularized HPV genomes

Support Protocol 3: Screening of fractions for viral proteins

Support Protocol 4: Screening of fractions for viral DNA

Support Protocol 5: Measuring viral titer

Support Protocol 6: Quantitation of quasivirions

Basic Protocol 2: Infection of primary human foreskin keratinocytes with quasivirus

Basic Protocol 3: HPV quasivirus transcription assay

Basic Protocol 4: HPV quasivirus replication assay

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人乳头瘤病毒准病毒的产生和原代人角质形成细胞的感染

本方案描述了人乳头瘤病毒(HPV)衍生的准病毒的生产。准病毒是在293TT包装细胞中产生的感染性颗粒，含有完整的病毒基因组。我们描述了用HPV准病毒感染原代人角质形成细胞的方法，以及测量早期病毒DNA复制和转录的方法。2020年出版。美国政府。基本方案1:转染，收获和分离HPV准病毒替代方案1:包装在293TT细胞中复制的HPV DNA替代方案2:使用“Ripcord”方法生产高纯度的准病毒支持方案1:生产HPV小环支持方案2:生产循环的HPV基因组支持方案3:筛选病毒蛋白的组分支持方案4:筛选病毒DNA的组分支持方案5:测定病毒滴度支持方案6:准病毒定量基本方案2:准病毒感染原代人包皮角质形成细胞基本方案3:HPV准病毒转录测定基本方案4:HPV准病毒复制测定

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来源期刊

Current Protocols in Microbiology Immunology and Microbiology-Parasitology

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期刊介绍： Current Protocols in Microbiology provides detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans and other microbes. It offers updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.