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The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product.

ABSTRACT: High-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties. To express the membrane (M) protein of SARS-CoV at high-level in vitro, the M gene fragment was amplified and cloned it into the Pichia Pastoris expression vector pPICZalphaA. SDS-PAGE and Western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of M protein was achieved, and that the expression product was similar antigenically to the natural protein. Purified recombinant M protein was used subsequently as an ELISA antigen for detection of eight serum samples screened previously by whole virus ELISA and immunofluorescence assay, and consistent results were obtained. These findings suggest that the recombinant M protein may be useful as a diagnostic reagent.

SUBMITTER: Han X

PROVIDER: S-EPMC7112794 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product.

Han Xueqing X Bartlam Mark M Jin Ying-Hua YH Liu Xiangtao X He Xiaojing X Cai Xuepeng X Xie Qingqe Q Rao Zihe Z

Journal of virological methods 20041201 1

High-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties. To express the membrane (M) protein of SARS-CoV at high-level in vitro, the M gene fragment was amplified and cloned it into the Pichia Pastoris expression vector pPICZalphaA. SDS-PAGE and Western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of M protein was achieved, and ...[more]

PMID: 15488627

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Project description:The diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection relies on the detection of viral RNA by real-time reverse transcription polymerase chain reaction (rRT-PCR) performed with respiratory specimens, especially nasopharyngeal swabs. However, this procedure requires specialized medical personnel, centralized laboratory facilities, and time to provide results (from several hours up to 1 d). In addition, there is a non-negligible risk of viral transmission for the operator who performs the procedure. For these reasons, several studies have suggested the use of other body fluids, including saliva, for the detection of SARS-CoV-2. The use of saliva as a diagnostic specimen has numerous advantages: it is easily self-collected by the patient with almost no discomfort, it does not require specialized health care personnel for its management, and it reduces the risks for the operator. In the past few months, several scientific papers, media, and companies have announced the development of new salivary tests to detect SARS-CoV-2 infection. Posterior oropharyngeal saliva should be distinguished from oral saliva, since the former is a part of respiratory secretions, while the latter is produced by the salivary glands, which are outside the respiratory tract. Saliva can be analyzed through standard (rRT-PCR) or rapid molecular biology tests (direct rRT-PCR without extraction), although, in a hospital setting, these procedures may be performed only in addition to nasopharyngeal swabs to minimize the incidence of false-negative results. Conversely, the promising role of saliva in the diagnosis of SARS-CoV-2 infection is highlighted by the emergence of point-of-care technologies and, most important, point-of-need devices. Indeed, these devices can be directly used in workplaces, airports, schools, cinemas, and shopping centers. An example is the recently described Rapid Salivary Test, an antigen test based on the lateral flow assay, which detects the presence of the virus by identifying the spike protein in the saliva within a few minutes.

Dataset Information

The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product.

Publications

The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product.

Similar Datasets

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