The Preparation Of Human Parvovirus B19 Virus-Like Particles

The parvovirus B19 is an erythrovirus included in the family of Parvoviridae. In healthy immunocompetent children, B19 is the cause of erythema infectiosum (also called the fifth disease), an innocuous rash illness. When infection occurs in adults, it will appear with acute symmetric polyarthropathy and transient aplastic crisis. In the immunocompromised host, infection with B19 will manifest as pure red cell aplasia or chronic anemia. Likewise, the pregnant woman who was infected with B19 would subject to hydrops fetalis or fetal death in utero. Transmission of B19 occurs via the respiratory route, through blood-derived products administered parenterally, or vertically from mother to fetus.It does make sense to develop a serological detection method of B19 since related methods are still absent in China. B19 IgG enzyme immunoassay can be utilized to discover previous infection. It's necessary to detect the content of IgG antibody against B19 in the donor's blood before being transfused to the pregnant women, as B19 IgG-positive blood seldom contain B19 virions in high titer. Moreover, the detection of B19 IgM is the best method of diagnosing an acute infection on a single serum sample.Sufficient viral antigens are needed to establish the ELISA method to determine the antibody against B19. However, the B19 virus is hard to cultivate in vitro and can not be the source of viral antigen. Therefore, the B19 antigen is generally produced by genetic engineering techniques. The B19 capsid comprises two kinds of capsomer proteins, designated VP1 and VP2. VP2 is the major capsid protein, which accounts for about 96% of the total proteins. In mammalian or insect cells, exogenetically expressed VP2 can self-assemble as virus-like particles (VLPs). The VLPs have proved to be the perfect antigen for antibody detection as they can efficiently bind to the conformational antibody.In this study, we aimed to produce B19 VLPs by over-expression of VP2 in prokaryotic and eukaryotic cells, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.1 Preparation of B19 VLPs by expression of VP2 protein in Sf9 insect cellsObjective: To prepare VLPs of B19 via expressing the VP2 protein in insect cells. Methods: The coding sequence of VP2 gene was synthesized by PCR-based two-step DNA synthesis method (PTDS). The PCR product was then cloned into the pMD18-T vector to generate pMD-VP2. After being confirmed by sequencing, VP2 was inserted into the multi-cloning site (MCS) of pFastBacl vector; the generated plasmid was transferred to the E.coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid) to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. The titer of the rBac-VP2 was determined by the method similar to the adenovirus, and then expressed VP2 protein by infecting the Sf9 insect cells using the rBac-VP2 at a multiplicity of infection of 10 PFU per cell. Indirect immunofluorescence and Western blotting were employed to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. Results: Bacmid carrying VP2 gene was generated using the baculovirus expression system. Recombinant baculoviruses were packaged in Sf9 cells after Bacmid-VP2 transfection, and the titer of rBac-VP2 was about 2.5×10~7 IU/ml. The expressed VP2 protein could be assembled to form VLPs in rBac-VP2-infected Sf9 cells as being identified by electron microscopy. Conclusions: The B19 VLPs were successfully produced in insect cells with baculovirus expression system. 2 Expression of B19 VP2 protein in prokaryotic cellsObjective: To prepare VLPs of B19 via expressing the VP2 protein in prokaryotic cells. Methods: The VP2 gene was subcloned into pET-30a(+) to generate pET-VP2 and pET-HVP2 by using different cloning strategies. VP2 or His-tag-fused VP2 were expressed in pET-VP2 or pET-HVP2 transformed BL21(DE3) cells by adding of the inducer of isopropyl-β-D-thiogalactoside (IPTG). In addition, the pBV-VP2 plasmid was constructed by inserting the VP2 gene to the pBV220 vector, and the protein expression was induced by changing the culture temperature. Ion exchange chromatography (IEX) and immobilized metal ion affinity chromatography (IMAC) were employed to purify the VP2 protein. The methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to evaluate the outcome of purification. The transmission electronic microscope (TEM) was utilized to observe the VLPs. Results: pET-VP2, pET-HVP2 and pBV-VP2 plasmids were successfully constructed. The results of SDS-PAGE indicated that VP2 protein was expressed in the form of inclusion bodies. The efforts of VP2 purification were fruitless since several peptides with a lower molecular weight than that of VP2 could not be excluded. These peptides could specifically bind the antibody against VP2 in western blotting, suggesting they were truncated VP2 peptides. VLPs were found to be absent in primarily purified VP2 under TEM. Conclusions: The VP2 protein could be successfully expressed as inclusion bodies in E.coli cells. However, it could hardly be purified due to the interfering of concomitantly-expressed truncated VP2. VLPs failed to be assembled in vitro in the system containing unpurified VP2 protein.In conclusion, we synthesized the VP2 gene firstly and cloned it to the prokaryotic expression vector and baculovirus expression vector, respectively. The VP2 protein was expressed in the E.coli and the Sf9 insect cells. Furthermore, The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.