Isolation and characteristics of virus culture of porcine epidemic diarrhea virus LJB/03 / 病毒学报

Porcine epidemic diarrhea virus (PEDV) LJB/03 strain was isolated from the feces of piglets suspected to be suffering from a severe diarrhea in Heilongjiang Province, and was identified by immunofluorescence test, immunelectronmicroscopy, RT-PCR and indirect ELISA assay. Characteristics of the virus culture and the methods of improvement of virus titer were explored. The results showed that the virus had the typical appearance of the coronavirus. Analysis of the nucleotide sequences of RT-PCR products revealed 98% homology with the reference strains. Indirect immunofluorescence assay showed a significant presence of green fluorescence, and an average P/N ratio of 7.6 by indirect ELISA assay. Taken together, these tests showed positive isolation of PEDV. Using the virus plaque purification cloning methods established in the test, the purified PEDV large plaque and small plaque were obtained, and the large plaque and small plaque titers were measured with significant difference. These results provide potential for the application of PEDV on the basis of the biological features of isolated virus.

Co-expression of PPV VP2 and LTB on the Surface of Lactobacillus casei / 微生物学通报

Lactobacillus casei was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant LTB and porcine parvovirus (PPV) VP2 protein. The fusion protein gene encoding PPV VP2 protein and LTB, was cloned into the surface expression vector pPG, and then the recombinant expression vector pPG-VP2-LTB was electrotransformed into Lactobacillus casei 393, generating recombinant strain pPG-VP2-LTB/L. casei 393. After induced by 2% Lactose in MRS broth, an about 78 kD protein was detected in the recombinant Lactobacillus casei by SDS-PAGE. The result of Western blot indicated that the protein possessed the antigenic specificity same as the native virus protein. The result of the whole bacteria cell ELISA indicated that the LTB protein was expressed at the same time. The results of indirect immunofluorescence test and immuno-gold electron microscopy showed that the interest protein was expressed on the surface of L. casei 393. The results provide potential for the development of lactic acid bacteria oral vaccine of PPV, which used LTB as mucosal adjuvant.

The surface display of porcine parvovirus VP2 protein in Lactobacillus casei / 生物工程学报

Lactobacillus casei 393 was selected as a bacterial carrier for the expression of Porcine Parvovirus (PPV) protective antigen VP2 protein. The gene encoding PPV VP2 protein was cloned into the Lactobacillus casei surface expression vector pPG, and then the constructed recombinant vector pPG-VP2 was electrotransformed into Lactobacillus casei 393 generating the recombinant system pPG-VP2/L. casei393 expressing PPV VP2 protein. The recombinant strain was induced by 2% Lactose in MRS and about 74kD protein was detected with SDS-PAGE. The result of Western-blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescent test showed that the expressed protein was secreted on the cell surface Lactobacillus casei.