J Vet Sci.  2017 Jun;18(2):159-167. 10.4142/jvs.2017.18.2.159.

Immunogenicity of recombinant Lactobacillus plantarum NC8 expressing goose parvovirus VP2 gene in BALB/c mice

Affiliations
  • 1College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University, Changchun 130118, China. yangguilian@jlau.edu.cn, wangchunfeng@jlau.edu.cn
  • 2Shandong Baolai-leelai Bioengineering Co. Ltd, Taian 271000, China.

Abstract

Goose parvovirus (GPV) continues to be a threat to goose farms and has significant economic effects on the production of geese. Current commercially available vaccines only rarely prevent GPV infection. In our study, Lactobacillus (L.) plantarum NC8 was selected as a vector to express the VP2 gene of GPV, and recombinant L. plantarum pSIP409-VP2/NC8 was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 70 kDa. Mice were immunized with a 2 × 109 colony-forming unit/200 µL dose of the recombinant L. plantarum strain, and the ratios and numbers of CD11c⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, and interferon gamma- and tumor necrosis factor alpha-expressing spleen lymphocytes in the pSIP409-VP2/NC8 group were higher than those in the control groups. In addition, we assessed the capacity of L. plantarum SIP409-VP2/NC8 to induce secretory IgA production. We conclude that administered pSIP409-VP2/NC8 leads to relatively extensive cellular responses. This study provides information on GPV infection and offers a clear framework of options available for GPV control strategies.

Keyword

Lactobacillus plantarum; VP2 gene; goose parvovirus; immunization

MeSH Terms

Animals
Female
Genes, Viral/genetics
Immunity, Cellular/immunology
Immunoglobulin A, Secretory/immunology
Lactobacillus plantarum/genetics/*immunology/virology
Mice
Mice, Inbred BALB C
Organisms, Genetically Modified
Parvoviridae Infections/prevention & control/veterinary
Parvovirinae/*genetics
Recombinant Proteins/immunology
Vaccines, Synthetic/immunology/pharmacology
Viral Proteins/genetics/*immunology
Viral Vaccines/immunology/pharmacology
Immunoglobulin A, Secretory
Recombinant Proteins
Vaccines, Synthetic
Viral Proteins
Viral Vaccines

Figure

  • Fig. 1 Polymerase chain reaction (PCR) results for the goose parvovirus (GPV) VP2 gene. M, marker DL2000; Lanes 1 and 2, PCR products of the VP2 gene (1764 bp); Lane 3, negative control.

  • Fig. 2 Confirmation of the pGEM-T-VP2 recombinant plasmid obtained via restriction enzyme digestion. Lane 1–4, pGEM-T-VP2 digested with KpnI and XbaI (3000 bp and 1764 bp, respectively); M1, DL2000 marker; M2, λ-Hind III.

  • Fig. 3 Electropherogram of the VP2 gene amplified via polymerase chain reaction (PCR) from the pSIP-409-VP2 recombinant plasmid. Lane 1, negative control; Lanes 2 and 3, VP2 PCR product (1764 bp); M, DL2000 marker.

  • Fig. 4 Confirmation of the pSIP-409-VP2 recombinant plasmid obtained via restriction enzyme digestion. Lanes 1 and 2, pSIP409-VP2 digested with KpnI and XbaI (5627 bp and 1764 bp, respectively); M1, λ-Hind III; M2, DL2000 marker.

  • Fig. 5 SDS-PAGE and western blotting analyses of the VP2 protein expressed in NC8. (A) All of the pSIP-409-VP2 protein bands at the induction times were 70 kDa in size. Lane M, protein marker; Lanes 1–6, induction of pSIP-409-VP2 with SppIP at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively; Lane C, empty pSIP-409 vector. (B) Western blotting analysis using an anti-GPV polyclonal antibody. The analysis demonstrates the specific reactivity of the VP2 protein expressed in NC8; the protein band was 70 kDa in size. Lane M, protein molecular mass standards; Lane 1, pSIP-409-VP2/NC8; Lane C, pSIP-409/NC8.

  • Fig. 6 CD11c, CD4, and CD8 expressions in immunized mice. Group A, pSIP409-VP2/NC8; Group B, pSIP409/NC8; Group C, phosphate-buffered saline (PBS). (A) Histograms show flow cytometry result (on isolated cells from spleen) from mice administered pSIP409-VP2/NC8 (thick red line) in comparison with the mice administered pSIP409/NC8 (thick green line) or PBS (filled histograms with gray color). (B) The CD11c+ levels of Group A were significantly different from those of Group B (*p < 0.05). (C) Gate strategy. (D) There were significantly different percentages of CD3+CD4+ and CD3+CD8+ cells between Groups A, B and C (**p < 0.01). On average, there were greater changes in CD11c+-, CD4+-, and CD8+-expressing spleen lymphocytes in Group A than in the other test groups. The data are expressed as mean ± SEM values from triplicate experiments. n = 3 mice per group.

  • Fig. 7 Effect of recombinant Lactobacillus plantarum on cytokine production in mice. The levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the supernatants of spleen lymphocyte cultures were determined by enzyme-linked immunosorbent assay. Values are the means ± SEM for three mice. Asterisks represent statistically significant differences; *p < 0.05, **p < 0.01 compared with the control group.

  • Fig. 8 Effect of administering recombinant Lactobacillus plantarum on sIgA production in mouse intestine. The levels of sIgA in the intestine were determined by enzyme-linked immunosorbent assay. Asterisks represent statistically significant differences; ***p < 0.001 compared with the control group. The data are expressed as mean ± SEM values of triplicate experiments. n = 3 mice per group.


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