The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli

Abstract

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones. Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein. A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29,459, was identified. The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E. coli of a chimaeric protein comprising most of the ORF encoding the Mr 29,459 polypeptide and beta-galactosidase. The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein. Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity.

The cloning and sequencing of the virion protein genes from a British isolate of porcine respiratory coronavirus: comparison with transmissible gastroenteritis virus genes

“…3). A similar M protein membrane signal sequence was identified in the TGEV Britton et al, 1988b) and on the M protein from the antigenically related coronavirus feline infectious peritonitis virus (FIPV; Vennema et al, 1991).…”

“…The position of oligo 60 is not shown as it corresponded to a region within PRCV ORF-3, not shown in this figure, described by Page et al (1991). Amino acid substitutions found on the TGEV FS772/70 (Britton et al, 1988a;1988b) and Purdue-115 The observation that the PRCV fragment A was about 600 bp smaller than the equivalent TGEV fragment would account for the observed difference in the size of PRCV mRNA 2 Page et al, 1991). The PRCV cDNA fragments A to H were cloned into pUC13 and the corresponding plasmids pPR137-7 (A), pPR137-9 (B), pPR137-11 (Cl, pPR137-13 (D), pKP-1 (El, pPR137-5 (F), pPR137-1 (G) and pPR137-3 (HI were used for DNA sequencing.…”

“…Oligonucleotides used for PCR amplifications were synthesised by the phosphoramidite method on an Applied Biosystem 381A DNA synthesizer. These were derived from published TGEV sequence data (Britton et al, 1988a;1988b;1989;Page et al, 1990; and are listed in Table 1. Fig.…”

A Serodiagnostic ELISA Using Recombinant Antigen of Swine Transmissible Gastroenteritis Virus Nucleoprotein.

“…X60056). Besides a single infection with PRCV, pigs are often exposed to combined infections with TGEV and PRCV [1,4]. Therefore, once seropositive animals are detected by the simple screening method described here, further differentiating diagnosis such as monoclonal antibody-based blocking ELISA [8] and spike gene targeted PCR should be applied.…”

“…In this study, we amplified the N protein coding sequence by RT-PCR using a set of specific primers with a NheI recognition sequence at the 5'-ends and genomic RNA of Japanese virulent strain, TO14 as a template [2]. The virus was propagated on porcine kidney (CPK) cells, harvested and purified by 35/50% discontinuous sucrose gradient ultracentrifugation as described previously [1]. Purified viruses at the interface were collected, then, used for genomic RNA extraction [5] or solublized with 1% Triton X-100 for ELISA antigen preparation [3].…”