title: [Construction and immunogenicity of recombinant porcine parvovirus-like particles with somatostatin].
abstract: In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.
authors.affiliation: National Research Center of Veterinary Biological Engineering and Technology, Jiangsu Academy ofAgricultural Sciences, Nanjing 210014, China.
authors.forename: Xuehua
authors.initials: X
authors.lastname: Zhang
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zheng
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.forename: Gang
authors.initials: G
authors.lastname: Xue
authors.forename: Hongyan
authors.initials: H
authors.lastname: Hou
authors.forename: Jibo
authors.initials: J
authors.lastname: Hou
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics*; D019677:Artificial Gene Fusion; D016367:Baculoviridae / Q000235:genetics; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D010322:Parvoviridae Infections / Q000517:prevention & control; D028323:Parvovirus, Porcine / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013004:Somatostatin / Q000235:genetics*; D013552:Swine; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis / Q000276:immunology*; D014765:Viral Vaccines / Q000096:biosynthesis / Q000276:immunology; D014771:Virion / Q000235:genetics / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C528774:VP2 protein, porcine parvovirus; D058425:Vaccines, Virus-Like Particle; D014765:Viral Vaccines; D013004:Somatostatin