Establishment Of ELISA,PCR For Detection TGE And Sequencing-Homology Analysis Of Partial S Gene

An indirect enzyme-linked immunosorbent assay(indirect ELISA)for detecting serum antibodies to the transmissible gastroenteritis virus (TGEV)was established by using cell culture grown TGEV as antigen for coating.This ELISA is a serological tool for TGE virus antibody and applied with advantage to testing large numbers of serum for the quarantine of import or export.335 serum samples were detected by the ELISA and by the serum virus neutralization test(NT),the result suggested the two methods' s qualitative agreement is 94.40%.The ELISA has the advantages of simplicity.repeatability.sensitivi-ty and rapidity.thus improves quarantine efficiency.According to S gene of TGEV, PRCV sequence.two pairs of primers were synthesized stretching S gene B, C antigenic sites.A reverse transcription (RT)-nested polymerase chain reaction(PCR) with the two pairs of primers was established for the differential detection of TEGV and PRCV.The PCR products of TGEV and PRCV had molecular sizes of 886 and 214 base pairs.respectively.The RT-nested PCR was proved to be very sensitive,specific,simple and rapid for diagnosis.Three Chinese isolated strains(shandong, huadu, TH-98)were amplified by the RT-PCR,The amplifications were sequenced and analyzed in comparison with other homologous strains.which derived from US,British,Korean,Japan and Taiwan of China.Gene sequence comparison demonstrated that the shangdong, huadu and TH-98 share above 93% identities with US(Neb72-rt, purdue-15. Miller), British (96-133, Fs772-70), Korean (133, Hkt2), Japan (Tol4), Taiwan of China (Tfl) and their amino acid homology were above 89%.Four antigenic sites A, B, C and D of TGEV are located on the 2.2Kb of 5 ' terminal S gene.Site A is conservative in the S glycoprotein protein.Amino acid residues 97~144 and 48~52 are part of site B and C respectively.Site C of Shandong, huadu, TH-98 can be represented by the peptide 48-P/S-P-N-S-D-52,Serine at position 51 is identity with reported data.The 97 th aminoacid residues Trp was substituted by Ser in antigentic site B of huadu strain,meantime,the 100th amino acid residues Arg was substituted by Lys.But the amino acid residues that contribute to antigentic sites B of shandong, TH-98 did not change.According to the phylogenetic tree.we find that shandong strain appeared phylogenetically farther to huadu, TH-98 strain. This study provides a basis of thestudies for the new type vaccine and molecular diagnosis.