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.

Identifying protein epitopes recognized by monoclonal antibodies / 生物工程学报

To establish a method for identifying protein epitopes recognized by therapeutic monoclonal antibodies, the programmed death receptor-1 (PD-1) was selected as the target protein. Based on the alanine scanning strategy, a rapid expression method of antigen mutants combining site-directed mutagenesis with mammalian cell expression system was established, the conditions for eukaryotic expression element amplification and cell transfection expression were established. 150 PD-1 protein mutants were co-expressed, and the binding ability of these mutants to anti-PD-1 antibody Pembrolizumab was identified. The epitopes of Pembrolizumab were determined based on the binding ability of protein mutants to antibodies and combined with protein structure analysis, which was highly consistent with the reported crystal structure-based epitopes, indicating that this method is simple and accurate and can be used for epitope mapping of therapeutic monoclonal antibodies.

Evaluation of the Toxicity of Silver/Silica and Titanium Dioxide Particles in Mammalian Cells

ABSTRACT The increasing concern over the spread of diseases has lead to a high consumption of antimicrobial additives in the medical and industrial fields. Since these particles can lixiviate from loaded materials, the contact between this additive and mammalian cells can occur during manufacture, use and disposal of the products. Silver on fumed silica (AgNP_SiO2) and titanium dioxide (TiO2) can be used as antimicrobial additives that are applied in polymeric formulation. While these additives can inhibit bacteria, fungus and virus proliferation; they may also be harmful to humans. Standard toxicological studies were undertaken using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide), CBPI (cytokinesis-block proliferation index) and micronucleus assay using different sets of additive concentrations. The nanosize of the samples evaluated was confirmed by transmission electronic microscopy. No significant micronucleus frequency increase or cell viability reduction were observed with the exposure of L-929 murine fibroblast cells to AgNP_SiO2 and TiO2 particles at any of the tested concentrations. The non toxic effect of the analyzed particles can be explained by considering its agglomeration tendency, composition, and crystalline form. Further investigations should be done to understand the interference of agglomeration and how it affects the toxicological study.

Analysis of expression profile of mce operon genes (mce1, mce2, mce3 operon) in different Mycobacterium tuberculosis isolates at different growth phases.

Background & objectives: Mycobacterium tuberculosis (M. tuberculosis) has four homologous mammalian cell entry (mce) operons (mce1-4) that encode exported proteins and have a possible role in the virulence mechanism of this pathogen. The expression of mce operon is considered to be complex and not completely understood. Although expression of mce operon at different in vitro growth phases has been studied earlier, its expression in different M. tuberculosis isolates under different growth phases is not yet studied. The present preliminary study was conducted on a limited number of isolates to know the trend of expression pattern of mce operon genes in different M. tuberculosis isolates under different growth stages. Methods: In this study, we monitored the transcriptional profile of selected mce operon genes (mce1A, mce1D, mce2A, mce2D, mce3A, mce3C) in different M.tuberculosis isolates (MDR1, MDR2, and sensitive isolate) at early exponential and stationary phases using real-time quantitative PCR. Results: The expression ratio of all selected mce operon genes in all M. tuberculosis isolates was reduced at the initial phase and increased substantially at a later phase of growth. Higher expression of mce1 operon genes was found in all M. tuberculosis isolates as compared to other mce operon genes (mce2 and mce3 operons) at stationary growth phase. Interpretation & conclusions: The higher expression of mce operon genes at stationary phase (as compared to early exponential phase) suggested growth phase dependent expression of mce operon genes. This indicated that the mce operon genes might have a role in M. tuberculosis survival and adaptation on the onset of adverse condition like stationary phase. Identification of differentially expressed genes will add to our understanding of the bacilli involved in adaptation to different growth conditions.

Research advances of recombinant coagulation factor VII expression and synthesizing mechanism / 中国药科大学学报

Haemophilia is caused by lack of coagulation factor VIII or IX in patients′blood with inadequate hemostasis.Currently recombinant coagulation factor VII(rFVII)produced in different cells is used against clini-cal bleeding of haemophilia patients.To enhance the production and activity of rFVII;some eukaryotic cells such as baby hamster kidney(BHK);Chinese hamster ovary(CHO);insect cell and fish embryo;were used to express rFVII.Meanwhile;the effect of functional gene on the activity of rFVII and the limitation of rFVII production caused by post-translational modification were investigated by different methods.The role of rFVII in hemostasis;synthesis of rFVII in different eukaryotic cells and impact on production of post-translational modification are reviewed in this article.

An enhancer/promoter combination strengthens the expression of blood-coagulation factor VIII in non-viral expression vectors

We explored the potential of fusion of hepatic locus control region 1 (HCR-1) with HCR-2 to express B-domain-deleted human factor VIII (FVIII) in four cell lines. B-domain-deleted human FVIII expression was controlled by HCR-1/HCR-2, followed by liver specific and ubiquitous promoters. Chimera enhancer HCR-1/HCR-2, followed by cytomegalovirus (CMV) promoter, gave 2-fold more FVIII expression in all cell lines (105.6 ± 2.8 for Hek-293, 68.8 ± 3.8 for HepG2, 34.8 ± 1.3 for CHO, and 27.2 ± 1.6 ng-mL-1-106 cells-1 for L.N.) when compared to the vector with CMV alone (54.8 ± 3.3 for Hek-293, 32.4 ± 1.2 for HepG2, 18.6 ± 1.1 for CHO, and 10.1 ± 1.7 ng-mL-1-106 cells-1 for L.N.). Elongation factor 1-α gene and human CMV promoters were more efficient than the promoters from the human α-1-antitrypsin gene, and fviii was less efficient in hepatic cell lines. HCR-1/HCR-2, followed by strong promoters, increases FVIII expression in vitro. Our results underscore the importance of cis sequences for enhancing in vitro FVIII expression; this may be helpful for designing new strategies to improve heterologous expression systems.

Expression of DNA transferred into mammalian cells.

There are several methods to introduce purified DNA into mammalian cells. These include microinjection into the nuclei of the recipient cells and complexing the DNA with facilitating agents such as calcium-phosphate. After it enters the nucleus, the DNA is expressed in a large proportion of the cells. This expression is dependent upon cis-acting sequences that are recognized by the mammalian transcriptional and translational machinery. In a smaller proportion of cells, the exogenous DNA becomes covalently integrated into the host cell DNA at random sites. Non-selectable genes can be introduced into mammalian cells by ligating them to a selectable marker or mixing the DNA with carrier DNA containing a selectable marker. The DNA that is introduced into mammalian cells can be rescued for examination and analysis. These gene transfer methods have already proven to be useful in identification of sequences which are necessary for normal gene expression as well as gene regulation. In addition a number of genes have been isolated using gene transfer methods. DNA mediated gene transfer holds much promise to target genes to specific sites in the chromosomes, to understand mechanisms of mammalian development and cell differentiation and is expected to provide a method to produce important and novel gene products that may be used for diagnostic and therapeutic purposes.