Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Expression of Aminopeptidase N1(APN1),the Main Receptor Protein for Bacillus thuringiensis CrylA Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 （APN1） gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt.

Carboxyl Terminus Truncated Human Papillomavirus Type 58 L1 Protein Maintains Its Bioactivity and Ability to Form Virus-like Particles

Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing-HPV58L1 protein-were harvested and analyzed by SDS-PAGE and Western blot. The ProBond~TM purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.

Sequence analysis and functional study of the Han Nationality glial cell line-derived neurotrophic factor transcript

Objective: To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR and expressed by baculovirus expression system. Biological activity of the expressed product was measured by the primary culture of midbrain dopaminergic neurons. Results: There only existed the shorter GDNF transcript of 555 bp in the Han nationality. The secretory expression product of the shorter transcript in insect cells promoted the survival and differentiation of dopaminergic neurons. Conclusion: It is found that there is a 78 bp deletion in the Han nationality GDNF transcript compared with the reported 633 bp GDNF transcript. The 78 bp deletion does not affect the secretory expression and biological activity of GDNF mature protein.

Recombinant Human IgG antibodies against Human Cytomegalovirus

Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus （HCMV） infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of VH and VL and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-K-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles(VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane(prM) and envelope(E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.

Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori

The silkworm genome encodes three iron storage proteins or ferritins, FerlHCH, Fer2LCH, and Fer3HCH. Probing our EST library constructed from 1-day- old silkworm eggs revealed only Fer2LCH mRNA, which encoded for a protein with a predicted putative N-glycosylation site. Developmental and tissue expression analyses dur- ing embryogenesis revealed that Fer2LCH mRNA was abundant from 6 h to 6 days after oviposition. Transcriptional expression of Fer2LCH during the postembryonic stage is also high in the larval fat body and mid-gut, and then is upregulated in all pupal tissues tested. We found that Fer2LCHmRNA contains an iron-responsive element, suggesting this fer- ritin subunit is subject to translational control. Although ferritin expression has been shown to increase following immune challenge in other insects, the levels of Fer2LCH mRNA were not significantly induced following viral or bacterial infection ofBombyx mori. Using a baculovirus expression system we expressed recombinant BmFer2LCH protein, which was detectable in the cytoplasmic fraction, likely in a compartment of the secretory path- way, and was shown to undergo posttranslational modifications including N-glycosylation. In particular, rBmFer2LCH carbohydrate chains were composed ofmannose and GlcNAc. We suggest that Fer2LCH is important for iron homeostasis and maintaining normal organ function in silkworms.

Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody

Background Thyroid peroxidase （TPO） is an important autoantigen in Hashimoto＇s thyroiditis （HT）, and almost all epitopes are located in TPO ectodomain. The glycosylation of TPO might contribute to breaking self-tolerance, therefore, pudfied glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT. The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO. Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain. The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays （ELISAs）. The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs. Results TPO ectodomain was recovered from the culture media as a soluble protein, and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography. The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies. Fucose, sialic acid and galactose were all detected on the recombinant TPO ectodomain. Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid. Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.