High-yield transgenic mammalian expression system for generating virus-like particles

Mammalian protein expression system has the function of protein folding and post-translational modification which let the protein closer to the natural protein, so that to obtain the same biological activity with natural protein. Therefore, the Mammalian Cell Expression System is the most widely used in the development and production of recombinant protein drugs, particularly in the therapeutic monoclonal antibodies. Email:marketing@medicilon.com.cn web:www.medicilon.com

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

To generate VLPs as a SARS vaccine, technical challenges include mammalian post-translational modifications and correct folding of viral proteins, their delicate organization into a lipid envelope, and sustainable yield for practical usage. The SARS-S protein is deduced as a huge glycoprotein containing 1255 aa residues with 23 putative N-linked glycosylation sites, and at least 12 N-glycans have been identified . In SARS-CoV infected cells and purified virion, protein M contains one high-mannose type N-glycan (Voss, D. et al., 2006, FEBS Lett 580: 968-73). Thus, mammalian expression and cell culture-based approaches are of interest to the inventors to attain massive production of SARS-VLPs.

In one aspect, the present invention provides a method for generating virus-like particles (VLPs) of a mammalian-hosted virus, such as SARS-CoV, the method comprising:constructing a plasmid comprising a nucleotide sequence encoding a combination of at least two structural proteins of the virus;transfecting Vero cells with the plasmid; and expressing the viral structural proteins in the transfected cells to generate VLPs of the virus.

The method of the present invention is suitable for generating various mammalian-hosted viruses, including but not limited to arenaviruses, coronaviruses, hepadnaviruses, herpes viruses, orthomyxoviruses, paramyxoviruses, papovaviruses, parvoviruses, and retroviruses. In a preferred embodiment of the present invention, the mammalian-hosted virus is a coronavirus. More preferably, the mammalian-hosted virus is SARS-CoV.

The term “viral structural protein” or “structural protein of a virus” and equivalent terms as used herein refers to viral genome-encoded proteins that form the structure of a virus, including membrane glycoproteins and capsid proteins. The genome of a virus also encodes non-structural regulatory proteins involved in virus replication. For example, the structural proteins of a coronavirus comprise the M (membrane), E (envelope), S (spike) and N (nucleocapsid) proteins.

In an embodiment of the method used to generate SARS-VLPs according to the invention, the structural proteins to be expressed in transfected cells can be any combinations derived with the E, M, N and S proteins of SARS-CoV, such as, for example, M+E, M+E+S, M+S, N+M+E, N+M+E+S, and N+M+S. In a preferred embodiment, the combination of the structural proteins is M+E. Most preferably, the combination of the structural proteins is M+E+S.

The plasmid used in the present invention can be any plasmid or vector suitable for expressing heterologous proteins in mammalian cells. Many commercially available mammalian expression vectors can be readily used in the present invention, for example, the pcDNA™ series by Invitrogen Corporation.

To construct the recombinant plasmid used in the present invention, nucleotide sequences encoding a combination of the viral structural proteins can be grouped into one or more “expression cassettes” for controlled expression. As used herein, the term “expression cassette” refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a nucleotide sequence of interest in a host cell. The expression cassette can be incorporated into a plasmid or chromosome. Typically, the expression cassette portion of an expression vector includes, among other sequences, a nucleotide sequence to be transcribed, a promoter, and a poly-adenylation signal. In the present invention, the term “expression cassette” is used interchangeably with the term “transgene.”