Pseudorabies (PR) is an infectious disease caused by the pseudorabies virus (PRV), affecting various domesticated and wild animals. Since pigs are the only natural hosts of PRV, PR poses a serious threat to the pig farming industry. Currently, PR is primarily prevented through vaccination with inactivated vaccines or genetically modified attenuated live vaccines. Developing safe and effective genetically engineered vaccines would facilitate the eradication and control of PR. In this study, the PRV vaccine strain Bartha-K61 was used as the reference strain. The gB protein was expressed via the baculovirus-insect cell expression system. Non-denaturing gel electrophoresis confirmed that the gB protein could form a trimeric structure. The purified gB protein was used to immunize mice, and the immune effect was evaluated by a challenge test. The results showed that the gB antigen induced a strong immune response in mice, with the serum-neutralizing antibody titer above 1:70. The lymphocyte stimulation index reached more than 1.29, and the level of (interferon gamma, IFN-γ) release was higher than 100 pg/mL. After immunization, mice were challenged with the virus at a dose of 10⁴ TCID₅₀/mL, 200 μL per mouse, and the clinical protection rate was 100%. Immunohistochemistry, histopathological section, and tissue viral load results showed that the pathological damage and viral load in the gB-immunized group were significantly lower than those in the PBS group. In summary, the gB protein obtained in this study induced strong humoral and cellular immune responses in mice, laying a foundation for developing a recombinant gB protein subunit vaccine.
Animals ; Mice ; Baculoviridae/metabolism* ; Viral Envelope Proteins/biosynthesis* ; Herpesvirus 1, Suid/genetics* ; Pseudorabies/immunology* ; Swine ; Pseudorabies Vaccines/genetics* ; Antibodies, Viral/blood* ; Insecta/cytology* ; Mice, Inbred BALB C ; Female ; Viral Vaccines/immunology*

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryotic membrane protein structures have been obtained due to the technical challenges in the generation of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.
Animals ; Escherichia coli ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; HEK293 Cells ; Humans ; Insecta ; cytology ; genetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; metabolism ; Yeasts ; genetics

BACKGROUNDThyroid 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, purified 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.

METHODSBac-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.

RESULTSTPO 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.

CONCLUSIONSHigh Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.

Animals ; Antibodies, Monoclonal ; immunology ; Baculoviridae ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Glycosylation ; Humans ; Insecta ; cytology ; Iodide Peroxidase ; immunology ; Recombinant Proteins

OBJECTIVETo establish a method for the expression of glycogen synthase kinase 3β with high purity and biological activity.

METHODSE.coli expression system and baculovirus-insect cell expression system were used to produce the kinase, followed by purification using His-tag and GST-tag and determination of its purity and activity by SDS-PAGE and kinase reaction, respectively.

RESULTSGlycogen synthase kinase 3β produced from E.coli represented 54% of the total bacterial protein, as compared with 96% of the total protein from the insect cell system .Glycogen synthase kinase 3β produced from insect cell exhibited an one-fold higher biological activity than the protein obtained from E.coli.

CONCLUSIONSCompared with the protein from E.coli system, glycogen synthase kinase 3β from the insect cell expression system is endowed with a higher purity and bioactivity.

Animals ; Baculoviridae ; metabolism ; Escherichia coli ; metabolism ; Genetic Vectors ; Glycogen Synthase Kinase 3 ; biosynthesis ; isolation & purification ; Insecta ; cytology

Recombinant adeno-associated virus has been proven to be a promising gene delivery vector for human gene therapy with many advantages. Successful applications of recombinant adeno-associated virus vectors in preclinical and clinical human gene therapies make it become a demanded product. A well-established and large-scale production system is therefore required. Since wild type of adeno-associated virus was well characterized in 1989, progress has been made. Particularly, package system of recombinant adeno-associated virus has been developed to use insect cell instead of human cell. These advances in adeno-associated virus production will allow it to meet the demands of basic research and clinical applications. This review will focus on trends in packaging systems and development on a large scale of recombinant adeno-associated virus production.
Animals ; Biotechnology ; methods ; Dependovirus ; genetics ; metabolism ; physiology ; Genetic Therapy ; Genetic Vectors ; biosynthesis ; genetics ; Humans ; Insecta ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Virus Assembly

To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter.
Animals ; Baculoviridae ; genetics ; metabolism ; Circovirus ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Insecta ; cytology ; metabolism ; Open Reading Frames ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Swine ; Viral Proteins ; genetics ; metabolism ; Virion

Human calcitonin (hCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. Calcitonin has important physiological function in vivo. We describe the couple expression of a synthesized modified human calcitonin(hmCT) gene fused with glutathione-S-transferase and rat peptidylglycine alpha-amidation monooxygenase (PAM) in insect cells infected by recombinant baculovirus GSTCT/PAM. Using Western blotting against hmCT or rat PAM, the GSThmCT fusion protein had been identified as well as the PAM. Following affinity chromatography with glutathione agarose column, the GSThmCT fusion protein produced by insect cells was purified. The purified fusion protein was also interacted with antibody against hmCT. The couple expression of a modification enzyme and its substrate in eucaryotic expression system may be used for producing other biological activity peptides.
Animals ; Baculoviridae ; genetics ; Calcitonin ; biosynthesis ; genetics ; Cells, Cultured ; Gene Expression ; Glutathione Transferase ; genetics ; Humans ; Insecta ; cytology ; Mixed Function Oxygenases ; biosynthesis ; genetics ; Multienzyme Complexes ; biosynthesis ; genetics ; Rats ; Recombinant Fusion Proteins ; genetics

The regulation of foreign gene expression in Insect-Baculovirus Expression System is very complex. In this report, the effect of 5'-UTR in the expression of hGH gene in cultured Sf9 cells was examined. A 18 bp length in the end of 5'-UTR of hGH (human Growth Hormone, hGH) cDNA including a stem-loop structure was deleted by PCR. The truncated hGH cDNA, delta 1hGH was cloned in pFastBac1, named pFast-Bac-delta 1hGH. After transforming into E. coli. DH10Bac, which have a shuttle vetor-Bacmid, the delta 1hGH was integrated into Bacmid by site-specific transposition, and an expression vector, rBacmid-delta 1hGH DNA was acquired. By transfecting the cultured Sf9 cells with the recombinant expression vector DNA, pure recombinant virus, rAcV-Bac-delta 1hGH was obtained, and hGH gene was expressed. Immuno-blot and Chemiluminescent assay revealed that the expressed hGH had normal immunological activity, the amount of hGH expression level in Sf9 cell supernatant infected with rAcV-Bac-delta 1hGH containing the truncated 5'UTR was four to five times higher than that infected with rAcV-Bac-hGH.
5' Untranslated Regions ; genetics ; Animals ; Base Sequence ; Cells, Cultured ; DNA, Complementary ; genetics ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Human Growth Hormone ; genetics ; metabolism ; Humans ; Immunoblotting ; Insecta ; cytology ; genetics ; Recombinant Proteins ; isolation & purification ; metabolism