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Title: Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection

Author
item MENG, FANDAN - Northeast Agricultural University
item ZHAO, ZEPING - China Agricultural University
item LI, GUANGXING - Northeast Agricultural University
item SUO, SIQINGAOWA - Northeast Agricultural University
item SHI, NA - Northeast Agricultural University
item YIN, JIECHAO - Northeast Agricultural University
item Zarlenga, Dante
item REN, XIAOFENG - Northeast Agricultural University

Submitted to: Virus Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2011
Publication Date: 6/24/2011
Citation: Meng, F., Zhao, Z., Li, G., Suo, S., Shi, N., Yin, J., Zarlenga, D.S., Ren, X. 2011. Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection. Virus Genes. 43:335-341.

Interpretive Summary: Transmissible gastroenteritis virus (TGEV) is the etiological agent of transmissible gastroenteritis. Although all age groups of pigs are susceptible to TGEV infection, the mortality rate can reach as high as 100% in sero-negative suckling piglets. This virus consists of a positive-stranded RNA genome that contains 4 structural proteins: the spike (S) protein, the integral membrane protein, the minor envelope protein, and the nucleocapsid protein. Among these, the S protein is the major host target for generating neutralizing antibodies to the virus. In the current study, we cloned and expressed a truncated TGEV S protein containing 2 key antigenic sites and showed that this construct not only induces specific antibodies recognizing TGEV-infected eukaryotic cells, but can interfere with cell infection by the virus. In addition, rabbit antibodies to the recombinant protein were capable of neutralizing TGEV when the virus was pretreated with the immune serum. These data provide important information for developing a recombinant vaccine against this disease and can be used by both scientists and commercial interest groups for advancing this goal.

Technical Abstract: The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell receptors and therefore specific cell types. It is also an important immune target for the host in neutralizing the virus. Four antigenic sites A, B, C and D that reside near the N-terminal domain have been defined in the S protein. Of these, the region encoding antigenic sites A and to a lesser extent D, herein defined as S-AD, are most critical in eliciting host neutralizing antibodies. Herein, we enzymatically amplified, cloned and expressed the S-AD fragment from TGEV in the prokaryotic expression vector, pET-30a. Maximum protein expression was achieved at 30oC over a 5 hr period post-induction. Rabbit polyclonal antiserum was generated using recombinant S-AD (rS-AD) protein. In contrast to prior studies showing no activity with bacterially-produced S protein, results indicated that polyclonal serum recognized TGEV-infected cells and reduced infection by 100%. Furthermore, the truncated rS-AD peptide was able to bind to the surface of cells from swine testes in a competitive manner and completely inhibit viral infection.