[The 40-91 aa sequence of porcine epidemic diarrhea virus ORF3 protein is the key structural domain controlling its location in cytoplasm].

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy.

Development of an epitope-based competitive ELISA for the detection of antibodies against Tibetan peste des petits ruminants virus.

Aims: To develop an effective diagnostic kit, based on a competitive ELISA-based system (cELISA), for detecting serum antibody against peste des petits ruminants virus (PPRV).

Methods: Epitope peptides of the nucleocapsid (N) protein of Tibetan PPRV were synthesized chemically and injected into rabbits to prepare hyperimmune antisera. Test sera were incubated simultaneously with hyperimmune antisera and added to the wells of ELISA plates coated previously with recombinant N protein.

Methanol/sorbitol co-feeding induction enhanced porcine interferon-α production by P. pastoris associated with energy metabolism shift.

The production of porcine interferon-α (pIFN-α) by Pichia pastoris was largely enhanced when adopting sorbitol/methanol co-feeding induction strategy at 30 °C in a 10-L fermentor. Analysis of energy regeneration pattern and carbon metabolism revealed that major energy metabolism energizing pIFN-α synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle under the methanol/sorbitol co-feeding induction strategy. The sorbitol/methanol co-feeding induction strategy weakened formaldehyde dissimilatory pathway and repressed the accumulation of toxic metabolite-formaldehyde, reduced theoretical oxygen consumption rate and oxygen supply requirement, and increased energy/methanol utilization efficiency so that more methanol could be effectively used for pIFN-α synthesis.

Development of an indirect ELISA with artificially synthesized N protein of PPR virus.

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain.

[Quasispecies investigation on swine hepatitis E viruses].

The objective of current study was to investigate the quasispecies of hepatitis E virus in swine. The partial ORF2 region of HEV envelope gene from four swine HEV strains was amplified by RT-nested polymerase chain reaction (RT-nPCR). After cloning and transformation of PCR products, 20 positive clones of each HEV isolate were subject to sequencing and DNA analysis.

Full genomic sequence analysis of swine genotype 3 hepatitis E virus isolated from Shanghai.

The full genomic nucleotide sequence of a previously identified genotype 3 hepatitis E virus (HEV), strain SAAS-JDY5, was obtained using RT-PCR and rapid amplification of cDNA ends (RACE). The genome consisted of 7225 nucleotides, excluding a poly-A tail at the 3' terminus, and contained three open reading frames (ORFs), ORF-1, ORF-2 and ORF-3, encoding 1702, 660 and 113 amino acids, respectively. Phylogenetic analysis confirmed that SAAS-JDY5 belonged to genotype 3 HEV and was most closely related to the Japanese isolate wbJYG1 (AB222184).

[Generation of Chicken Germ-line Chimeras by Transferring PGCs and Their Identification by AFLP.].

PGCs (Primordial germ cells) were isolated from the blood of 51~56 h hatching Shiqiza chicken embryos by Ficoll density gradient centrifugation. The PGCs were injected into 2.5 d hatching embryos of H breed chicken to produce germ-line chimeras.

[High level secretion expression of PoIFNalpha in Pichia pastoris].

The porcine alpha interferon gene was inserted into the Pichia pastoris expression vector of pPICZalphaA which contains AOX I promoter and alpha-factor signal sequence. The recombinant plasmid was transformed into host cell E.coli JM109 and then was extracted for analysis of restriction enzymes.

[Site-directed mutation of PoIFN-alpha and its expression in Escherichia coli].

By using huge primer PCR Cys86 (TGC) of PoIFN-alpha was mutated to Tyr(TAC), and the first code TGT was simultaneously changed to TGC, which is a bias code of E. coli. The expression plasmid pGEX-IFN was constructed successfully.