Construction and immunogenicity of recombinant porcine parvovirus-like particles with somatostatin.
- Author:
Xuehua ZHANG
1
;
Qisheng ZHENG
;
Jin CHEN
;
Gang XUE
;
Hongyan HOU
;
Jibo HOU
Author Information
1. National Research Center of Veterinary Biological Engineering and Technology, Jiangsu Academy ofAgricultural Sciences, Nanjing 210014, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antigens, Viral;
biosynthesis;
genetics;
Artificial Gene Fusion;
Baculoviridae;
genetics;
Capsid Proteins;
biosynthesis;
genetics;
Mice;
Parvoviridae Infections;
prevention & control;
Parvovirus, Porcine;
genetics;
immunology;
Recombinant Proteins;
biosynthesis;
genetics;
immunology;
Somatostatin;
genetics;
Swine;
Vaccines, Virus-Like Particle;
biosynthesis;
immunology;
Viral Vaccines;
biosynthesis;
immunology;
Virion;
genetics;
immunology
- From:
Chinese Journal of Biotechnology
2010;26(8):1057-1067
- CountryChina
- Language:Chinese
-
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.
