3.The structure and virus-like particle vaccine of the HIV-1 capsid protein.
Xiao-Guang SUN ; Chang-Zheng SONG
Chinese Journal of Biotechnology 2003;19(5):516-520
The HIV-1 capsid protein (CA) plays an essential role in viral core assembly and maturation. Proteolytic cleavage at the MA-CA junction of the retroviral gag polyprotein refolds the amino-terminal end of capsid into a beta-helix structure that is stabilized by a salt bridge between the protein's processed amino-terminus and a conserved acidic residue. The refolded capsid aminoterminus then creates a new CA-CA interface, allowing assembly of the mature capsid core. Recently, researches focus on assembly of CA in vitro and development of CA vaccine. CA vaccine will provide widely immune protection because CA is comparatively conserved. Experiments demonstrate that fusing as few as four matrix residues onto the amino-terminus of capsid redirects protein assembly from cylinder to spheres in vitro. Evaluation of immunogenicity showed that immunization with virus-like particles induced both cellular and neutralizing antibody responses. Furthermore, mucosal administration of virus-like particles effectively induced both mucosal and systemic immune responses. These results indicate that virus-like particles consisting of HIV structural proteins are an attractive vaccine platform for eliciting anti-viral immune responses, especially neutralizing antibody responses. The production of antigens for vaccines in plants indicates that plant-based transgenic expression represents a viable means of producing CA vaccine for the development of HIV vaccine and for use in HIV diagnostic procedures and it has the potential as a safe and cost-effective alternative to traditional production systems.
AIDS Vaccines
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immunology
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Capsid
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immunology
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metabolism
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Capsid Proteins
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genetics
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immunology
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metabolism
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HIV-1
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genetics
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immunology
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metabolism
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Virion
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genetics
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immunology
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metabolism
4.Simple Purification of Adeno-Associated Virus-DJ for Liver-Specific Gene Expression.
Yonsei Medical Journal 2016;57(3):790-794
Recombinant gene expression using adeno-associated viruses (AAVs) has become a valuable tool in animal studies, as they mediate safe expression of transduced genes for several months. The liver is a major organ of metabolism, and liver-specific expression of a gene can be an invaluable tool for metabolic studies. AAV-DJ is a recombinant AAV generated by the gene shuffling of various AAV serotypes and shares characteristics of AAV2 and AAV8. AAV-DJ contains a heparin-binding domain in its capsid, which suggests that a heparin column could be used for the purification of the AAV. Given that AAV-DJ has been only recently available, relatively little is known about the optimal preparation/purification and application of AAV-DJ. Here, we present a simple large-scale preparation method that can generate 3×10(13) viral particles for in vivo experiments and demonstrate liver-specific gene expression via systemic injection in mice.
Animals
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Capsid
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Capsid Proteins/*genetics
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Dependovirus/*genetics
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*Gene Expression
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*Genetic Vectors
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Genome, Viral/genetics
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Hep G2 Cells
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Humans
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Liver/*metabolism
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Mice
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Mice, Inbred C57BL
5.Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris.
Ping-chuan LI ; Xiao-guang ZHANG ; Ling ZHOU ; Yi ZENG
Chinese Journal of Experimental and Clinical Virology 2003;17(4):310-314
OBJECTIVEHuman papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.
METHODSThe whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.
RESULTSThree clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.
CONCLUSIONHPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.
Capsid Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Genes ; Papillomaviridae ; genetics ; Pichia ; genetics ; metabolism ; Viral Proteins
6.Expression and purification of human papillomavirus type16 L1 protein in a prokaryotic expression system.
Guangyu BAO ; Hongxi GU ; Daohong LIN ; Min ZHUANG ; Lihua SHUI ; Jing WANG
Journal of Biomedical Engineering 2002;19(2):280-283
This study was intended to establish a method of purification of HPV16 L1 protein expressed in a prokaryotic system and to obtain the purified protein. The prokaryotic expression vector pGEX-4T-1-HPV16 L1 was constructed and transformed into E. coli BL21 cell, and induced by 1 mM IPTG to express HPV16L1 protein. The inclusion bodies were isolated and solubilized with 8 M urea. After the urea was removed by gradual dialysis, the denatured L1 protein were renatured and then were purified by affinity chromatography. The results showed that HPV16L1 protein formed inclusion bodies in bacterial expression system, suggesting that this assay can be used to purify HPV16L1 protein and hence provide a basis for studying the applications of HPV16 L1 protein.
Capsid Proteins
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biosynthesis
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Escherichia coli
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metabolism
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Genetic Vectors
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Human papillomavirus 16
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Oncogene Proteins, Viral
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biosynthesis
7.Construction and identification of attenuated Salmonella which harboring enterovirus 71 VP1 gene.
Ze LIU ; Yan HU ; Hong-hui SHEN ; Shao-ping CAI ; Bing-ke BAI ; Rong GAO ; Sheng-dong LUO ; Yan-tao CHAI ; Pan-yong MAO
Chinese Journal of Experimental and Clinical Virology 2011;25(2):117-119
OBJECTIVETo develop attenuated Salmonella which harboring enterovirus 71 (EV71) VP1 gene.
METHODSThe plasmid which expressed VP1 protein of EV71 was constructed by gene recombination. Cellular expression was assessed by Western Blot analysis. The recombinant plasmid was then transformed into attenuated Salmonella SL7207.
RESULTSEV71 VP1 gene sequence was inserted into a eukaryotic expression plasmid VR1012. VP1 protein was detected by Western Blot analysis in the culture supernatant. And the attenuated Salmonella harbored the plasmid stable.
CONCLUSIONThe plasmid was constructed successfully and it can express effectively in vitro. The bacteria which harboring the plasmid were constructed successfully. This has provided a basis for further research of an oral EV71 vaccine.
Capsid Proteins ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; Gene Expression ; Genetic Engineering ; Genetic Vectors ; genetics ; metabolism ; Salmonella ; genetics ; metabolism
8.Expression and assembly of rotavirus-like particles in insect cells mediated by recombinant Bombyx mori MultiBac.
Hu LONG ; Lun-guang YAO ; Shan-shan WANG ; Shin-xin CHEN ; Pei-chan TAN ; Jing-chen SUN
Journal of Southern Medical University 2010;30(7):1491-1495
OBJECTIVETo construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.
METHODSHuman group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.
RESULTSThree genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.
CONCLUSIONWe have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.
Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; virology ; Capsid Proteins ; genetics ; Cell Line ; Gene Expression ; Genetic Vectors ; Rotavirus ; genetics ; metabolism
9.A versatile building block: the structures and functions of negative-sense single-stranded RNA virus nucleocapsid proteins.
Yuna SUN ; Yu GUO ; Zhiyong LOU
Protein & Cell 2012;3(12):893-902
Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs.
Animals
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Capsid
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metabolism
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Humans
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Lassa virus
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chemistry
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physiology
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Nucleocapsid Proteins
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chemistry
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metabolism
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Orthobunyavirus
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chemistry
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physiology
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RNA Viruses
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chemistry
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physiology
10.Promotion of self-nucleic acid fragments on the assembly of foot-and-mouth disease virus-like particles.
Haiyun LIU ; Hu DONG ; Ye JIN ; Huichen GUO ; Shiqi SUN
Chinese Journal of Biotechnology 2020;36(10):2076-2082
The special nucleic acid fragments, 5' untranslated region (5' UTR) and internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV), which interact with the capsid proteins, were selected as scaffolds to investigate the assembly efficiency of foot-and-mouth disease (FMD) virus-like particles (VLPs). The assembled product was characterized by evaluation of particle size, surface potential, gel retardation assay, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. The results confirmed that the 5' UTR and IRES of FMDV co-assembled with the FMD VLPs and facilitated the assembly efficiency of FMD-VLPs. It demonstrates that the assembly efficiency of 75S particles of VLPs-5'UTR was significantly higher than those of the VLPs (P<0.001) and VLPs-IRES group (P<0.01). Comparatively the assembly efficiency of 12S particles of VLPs-IRES was significantly higher than those of the VLPs (P<0.000 1) and VLPs-5'UTR (P<0.000 1). It showed that the 5' UTR represented more effective in facilitating the assembly of VLPs. This study proposes an optimized strategy for improving the assembly efficiency of VLPs for the development of VLPs vaccine.
5' Untranslated Regions
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Capsid Proteins/metabolism*
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Foot-and-Mouth Disease Virus/physiology*
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Internal Ribosome Entry Sites
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Nucleic Acids/metabolism*
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Virus Assembly