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| Cloning and Expression Analysis of Nucleocapsid Protein-encoding Gene(N) from Porcine Transmissible gastroenteritis virus(TGEV) Fujian Strain |
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Abstract Transmissible gastroenteritis (TGE) is a coronavirus which causes enteric disease in swine of all ages. To investigate the sequence characteristic of the nucleocapsid protein gene(N) and its prokaryotic expression product of the porcine Transmissible gastroenteritis virus in Fujian Province(TGEV-FJ), the N gene was amplified by RT-PCR with primers designed according to TGEV strains in GenBank. The sequence analysis demonsrated that the N gene of TGEV-FJ strain was 1 149 bp encoding a protein of 382 amino acids(GenBank accession No. JQ700302), which shared high degree of identity for nucleotide and amino acid sequences with TGEV reference strains(95.4%to99.8% and 96.1%to100%, respectively). The N gene was cloned into pET-32a(+)-C vector for expression in E.coil Rosetta(DE3) after IPTG induction. SDS-PAGE analysis showed that the N product was 68 kD. Western blot demonstrated that the expressed recombinant protein was recognized specifically with porcine anti TGEV-FJ serum. An indirect ELISA coated with the purified recombinant nucleocapsid protein showed specificity for the detection of antibody against TGEV, 16 out of 20 sera from piglets(Sus scrofa) infected with TGEV were positive, and 9 out of 10 healthy sera were negative. Balb/c mice were immunized with the purified expression product of N protein. One hybridoma cell line (named 1-27)stably secreting McAbs against pET-32a-N was obtained, and the McAbs had strong reaction with TGEV antigen by indirect immunofluorescence assay (IFA). The McAbs prepared in this study can be served as a useful tool for developing immunological diagnostic techniques for TGEV infection.
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Received: 01 January 1900
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ZHU Xiao-Feng, XU Hou-Qiang, CHEN Wei, CHEN Ying-Lian, NI Kai, WU Xiao-Min, NI Meng-Meng, LU Xian-Jun. Cloning, Expression and Bioinformatics Analysis of IGF-1 and IGF-2 Genes in Congjiangxiang Pigs (Sus scrofa)[J]. 农业生物技术学报, 2019, 27(8): 1382-1391. |
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