Expression of influenza B virus hemagglutinin and its immunogenicity determination / 生物工程学报

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

Construction and application of recombinant human UDP-glucuronosyltransferases expression systems / 药学学报

In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, 1H NMR, and 13C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells / 中华微生物学和免疫学杂志

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells / 中华微生物学和免疫学杂志

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.

Characterization of N-glycosylation in an anti-EGFR monoclonal antibody produced by different expression systems / 生物工程学报

The use of mammalian expression systems results in a remarkable heterogeneity of mAb products, generally due to post-translational modifications, and glycosylation is a critical post-translation modification because it has a profound impact on the safety and efficacy of mAbs. The present study was designed to explore the impact of a different expression system on mAb N-glycosylation. The detailed structures of individual glycans between anti-EGFR monoclonal antibodies produced by different expression systems were successfully characterized at the level of free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry (LC-ESI-QTof MS). An alternating low and elevated collision energy scan, in source collision-induced dissociation and MS/MS in combination with exoglycosidase digestion method was also adopted. The combined data revealed that the Fab region of anti-EGFR antibody produced by CHO cell expression system had a pattern of glycosylation differing from that of the SP2/0 cell expression system whereas the Fc region remained basically unchanged. We confirmed that anti-EGFR antibody produced by SP2/0 cell expression system had a much more diverse mixture of glycans with α-Gal and an undesired, aberrant form of sialylation N-glycolylneuraminic acid (NGNA). The α-Gal was absent in mAb produced by CHO cell expression system containing sialic acid predominantly N-acetyl neuraminic acid (NANA) which is the desired, normal human-type sialylation. This study theoretically predicts that anti-EGFR antibody produced by CHO cell expression system may show better clinical tolerance, and very low potential for active hypersensitivity reactions, CHO cell lines can be the preferred expression system for producing anti-EGFR biobetter.

Characterization of HIPSTR highlights the heterogeneous expression pattern of lncRNAs in human embryos and stable cell lines / Caracterização do HIPSTR destaca o padrão de expressão heterogênea de IncRNAs em embriões humanos e linhagens estáveis de células

There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns

Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos

Effects of mechanical stimulation on basic fibroblast growth factor expression of corneal fibroblasts / 医用生物力学

Objective To investigate the effect of different mechanical environment ( in vivo and in vitro) on expression of basic fibroblast growth factor (bFGF) and explore the role of mechanical stimulation in corneal tissue repair after laser assisted in situ keratomileusis (LASIK) surgery. Methods Animal models by LASIK surgery were established to keep the corneas under different mechanical environment. The experimental animals were killed at the first week or the first month after LASIK surgery to obtain the corneas. In addition, the primary corneal fibroblasts were subjected to cyclic mechanical stretch (0.1 Hz; 5%, 10%, 15% stretch; 6 h or 24 h) using Flexcell 4 000 tension system. Expression of bFGF was determined by ELISA method. Results At the first week after LASIK surgery, expression of bFGF was increased significantly in 30% group (residual stroma bed accounting for 30% of the whole cornea), as compared with the control group (P<0.05), and then it was decreased to the normal level in all groups at the first month after LASIK surgery. Analysis on the same surgery method at different time showed that there were significant differences only in 30% group at the first week and month (P<0.05). Cyclic stretch experiment in vitro indicated that bFGF expression in 15% stretch group was significantly increased after 6 h than that in the control group (P<0.05), with a significant decrease after 24 h (P<0.05). Conclusions Mechanical stimulation can regulate bFGF expression of corneal tissues and corneal frbroblasts, and bFGF plays a positive role in the early corneal tissue repair after LASIK surgery.

Effects from cyclic mechanical stretch on proliferation of rheumatoid arthritis fibroblast-like synoviocytes / 医用生物力学

Objective To investigate the effects from cyclic mechanical stretch on proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). Methods In the experimental groups, cyclic mechanical stretch, with frequency of 1.0 Hz and magnitude of 3%, 6% and 9%, respectively, was applied to RA-FLSs for 2 h, 6 h, and 12 h. The control group remained in the same culture condition as the experimental groups, but without any mechanical stretch. After mechanical loading, the cell viability was analyzed by MTS, and its proliferation was assayed by flow cytometry. RT-PCR was used to measure the gene expression of the cell cycle regulatory factors, including CDK2, cyclinD1, cyclinE1, and P27. Results Cyclic mechanical stretch with magnitude of 6% and 9% for 6 h or 12 h significantly decreased the cell proliferation and viability in RA-FLSs (P0.05). Conclusions The effects from cyclic mechanical stretch on proliferation of RA-FLSs depend on the stretch magnitude and duration. Mechanical stretch with magnitude of 6% and 9% can inhibit RA-FLSs proliferation, which may be achieved by regulating the expression of Cyclin E1, CDK2 and P27. This study provides references for investigating the role of mechanical stimulation in pathogenesis of rheumatoid arthritis, as well as its prevention and treatment.

Construction and identification of pneumocytis carinii eukaryotic expression plasmids of pVAX-p55-v3 and pVAX-p55-v0 antigenic gene / 中国免疫学杂志

Objective:To construct eukaryotic expression plasmids of pneumocystis carinii p55-v3 and p55-v0 antigenic genes and to identify their expression in COS-7 cells at mRNA level.Methods:Pneumocystis carinii total RNA was used as the template to amplify p55-v3 and p55-v0 antigenic gene by RT-PCR.The products were connected to pTA2 vector and then cloned in pVAX1 eukaryotic expression vector to construct recombinant plasmids as pVAX-p55-v3 and pVAX-p55-v0.After propagated in E.coli DH5α,the recombinant plasmids were transfected into COS-7 cells.After 24 h incubation,the RT-PCR was performed to identify the mRNA expression of p55-v3 and p55-v0 antigenic gene.Results:The recombinant plasmids were qualified by restrictive endonuclease digestion and sequencing.And when compared with that in GenBank,the homology of p55-v3 antigenic gene was 99.9% in nucleotides and 100% in amino acid.The homology between p55-v0 antigenic gene and the one reported previously in nucleotide and amino acid seguence were 99.8% and 100%.The results of RT-PCR confirmed that p55-v3 and p55-v0 antigenic genes were transfected into COS-7 cells successfully and the genes were expressed in the cells.Conclusion:In this study,the recombinant plasmids of pVAX-p55-v3 and pVAX-p55-v0 are conducted successfully and expressed in the COS-7 cells,which provide a basis for clarification of immunologic function of p55-v3 and study of DNA vaccine.

Recombinant Baculoviruses as Mammalian Cell Gene-delivery Vectors:a Review / 中国生物工程杂志

The baculovirus expression system has been used extensively for the expression of recombinant proteins in insect cells.Recently,recombinant baculoviruses containing mammalian cell-active promoter element have been used to transduce a broad spectrum of primary and established mammalian cells,including non-hepatic cells.Recombinant baculoviruses have been used successfully for transient or stable gene delivery in mammalian cells in vitro,while the efficiency of delivering gene in vivo is inhibited obviously by complements,but efforts have been made to overcome the problems,for instance,VSV-G-pseudotyped baculoviruses display complement resistance.The mechanism of the transduction is still not clearly understood,though many researches have been done.The baculovirus is able to replicate only in insect cells,but it is incapable of initiating replication cycle in mammalian cells,which guarantees high biosafety of this gene delivery system.In addition,this system is easily manipulated and able to carry large inserts.These attributes will undoubtedly lead to the increased application and continued development of this system for highly effective gene delivery into mammalian cells.

Construction and expression of N-terminal mutants of human Eotaxin / 第三军医大学学报

Objective To construct prokaryotic cell expression vector of human Eotaxin mutants.Methods By point mutation,eight amino acid residues in the N-terminal(residues of 3-7)and N-loop(residue 14)regions of Eotaxin were individually mutated to methionine and residue 14 was delleted or methiomine was inserted after the residue 14,and then cloned respectively into prokaryotic cell expression vector-PET30a+.Results Eight N-terminal and N-loop mutants of human Eotaxin and their prokaryotic cell expression vector-PET30a+ were gained.Conclusion The successful construction of prokaryotic cell expression vector of human Eotaxin mutants lays a foundation for their expression and biological activity and for filtering antagonists of CCR3.

Transfection of osteoprotegerin gene into Cos-7 cells / 实用口腔医学杂志

Objective:To transfect human osteoprotegerin( o pg) gene into Cos-7 cells.Methods:The primers were designed b ased on the human opg cDNA sequence,total mRNA was isolated from 293 cells and RT-PCR was performed.The fragment of opg cDNA was inserted into pSecTag 2/B vector and sequenced by automatic sequence analyzer. The recombined plasmid was transfected into Cos-7 cells by Lipofectamine 2000 and OPG protein expressi on in Cos-7 cells was determined by immunohistochemistry and Western blot. Results:The sequence of opg cDNA from 293 cells obtained by RT- PCR was identical to the sequence provided by GenBank [gi:33878056]. The fragm ents of the recombinant plasmid digested with Hind Ⅲ,EcoR I and BamH Ⅰ and exa mined by 10 g/L agarose electrophoresis were consistent with predicted size.OPG over-expressing Cos-7 cells was selected and confirmed by immunohistochemistry and Western blot.Conclusion:Human opg gene may be transfect ed into Cos-7 cells.

The expression and its biolgic specification of the Ku80 gene molecular cloning / 中国免疫学杂志

Abstract Objective:To study the expression and its biologic specification of the Ku80 gene molecular cloning.Methods: Constructingrecombinant sense or antisense Ku80 expression vectors (Ku80/pCAGGS) and transferring into LCA .Those Ku80 mRNA and protein were de-teited in the Ku80s-LCA, Ku80as-LCA and by Northern-blotting and or Western-blotting.Results: Those Ku80 mRNA were detected in the Ku80s-LCA and Ku80as-LCA by Northern-blotting. Only the sense Ku80 protein was defected in the Ku80s-LCA , but wasn' t detected in the Ku80as-LCA by Western-blotting.Conclusion:The sense or antisense Ku80 were not only transferred in to LCA ,but also expressed in the mR-NA . Only the sense Ku80 protein was expressed in the Ku80s-LCA , but wasn' t expressed in the Ku80as-LCA . The Ku80 is a candidate tar-get for cancer gene therapy.

The expression of human fibroblast growth factor receptor 1 on the membrane of insect cell Sf9 / 中国免疫学杂志

Objective:Express the recombinant human FGFR1 in order to screen the FGFs.Methods:A cDNA fragment encoding human fibroblast growth factor receptor 1(FGFR1) was isolated by RT PCR from human lung fibroblasts,and then cloned into pCR TM II plasmid and pFastBac1 donor plasmid.Through transposition,recombinant bacmid FGFR1 DNA was formed and was then transfected into Sf9 insect cells to produce recombinant Baculovirus.Sf9 insect cell were infected with recombinant Baculovirus.The harvested culture supernatant was subjected to Western Blot and ELISA analysis.Results:The size of FGFR1 cDNA fragment is 2 100 bp.The MW of expressed recombinant FGFR1 was 78 kD.ELISA showed that human recombinant FGFR1 was expressed on the membrane of insect cell Sf9.Conclusion:The recombinant human FGFR1 can be expressed on the membrane of insect cell Sf9 effectively.

Construction of HCV NS3 epitope fusion gene vector and its expression in eukaryotic cell line / 解放军医学杂志

Objective To construct a fusion minigene expression vector of seven HCV NS3 epitopes and express the fusion gene in the eukaryotic cell line, in order to form a foundation for the investigation of the DNA immunization for HCV prevention. Methods Three pairs of complementary oligonucleotides primers covering all seven HCV NS3 epitopes were synthesized. Overlapping extension PCR were performed to construct the fusion minigene and cloned in pEGFP-N3 and pBuDCE vector respectively. The fusion protein was detected by Western blotting and flow cytometric analysis. The transfection efficiency was assessed with by flow cytometric analysis. Results The HCV NS3 epitope fusion minigene was obtained and inserted into pEGFP-N3 and pBuDCE vector respectively. The recombinant plasmid pEGFP-DR4 and pBuDCE-DR4 were expressed in 293T and 046W cell lines respectively. The Western blot result indicated that the expected 36 kDa minigene/EGFP fusion product was expressed from pEGFP-DR4, while 13kDa product from pBuDCE-DR4 respectively. Conclusion The HCV NS3 epitope fusion minigene was expressed in the eukaryocyte expression system.