Objective: To evaluate antigenic structure of multivalent epitope vaccine CWAE against Helicobacter pylori (H. pylori) by bioinformatics softs, obtain the purified CWAE protein after prokaryotic expression, and further identify the immunological properties of CWAE.Methods: The antigenic structure of CWAE was analyzed by bioinformatics softs. The recombinant plasmid pET28a-CWAE was obtained by using a synthetical WAE gene to replace the UE gene in pET28a-CUE plasmid. Then, the recombinant plasmid pET28a-CWAE was transformed into E. coli BL21 (DE3). After induction by IPTG, the antigen protein CWAE was purified by Ni-NTP nickel ion affinity chromatography. The adjuvant activity of CTB components in CWAE was identified by GM1-ELISA. Finally, the ability of CWAE to induce antibodies and lymphocyte immune response against H. pylori was detected by ELISA and spleen lymphocyte proliferation test.Methods: The multivalent epitope vaccine CWAE had a scientific and reasonable structure. The CWAE gene in recombinant plasmid pET28a-CWAE was consistent with the design sequence. After induction with IPTG, CWAE protein mainly exists as inclusion body. The purified CWAE was about 93.2% after purification.Results: from GM1-ELISA confirmed that CTB components in CWAE had good mucosal adjuvant activity. ELISA results confirmed that CWAE could stimulate BALB/c mice to produce anti-H. pylori antibodies. Moreover, CWAE vaccine could stimulate lymphocyte responses against various pathogenic factors from H. pylori.Conclusion: H. pylori multivalent epitope vaccine CWAE has scientific and reasonable antigen structure, and CWAE with high purity can be obtained by prokaryotic expression. Furthermore, the CWAE vaccine can stimulate specific antibodies and lymphocyte response against H. pylori. Experimental evidences for the development of multivalent epitope vaccine against H. pylori will be provided.
Objective: To explore more effective S. pneumoniae DNA vaccines and vaccine immunization strategies and explore their protective mechanisms.Methods: The recombinant plasmid pcDNA3-dnaJ was constructed and DnaJ protein was expressed. The recombinant plasmid pcDNA3-dnaJ/protein DnaJ immunized group and the plasmid pcDNA3-dnaJ immunized group were separately set to compare the nasal lavage of mice treated with S. pneumoniae strain. The bacterial load and survival rate of the liquid, serum antibody titer and inflammatory factors after challenged were measured by ELISA. The activation of BMDCs and the immune responses of Th1 and Th17 cells were analyzed by flow cytometry.Methods: The plasmid pcDNA3-dnaJ immunization three times induced antigen-specific antibody in serum and reduced the bacterial loads in the nasopharynx after challenge with live S. pneumoniae, but it was less effective in protecting against a lethal infection. However, compared with repeating the plasmid DNA innoculation three times, the strategy of pcDNA3-dnaJ prime one time/DnaJ protein boost one time could significantly reduce the pneumococcal colonization in the nasopharynx and provid better protection against lethal infection. Furthermore, DnaJ protein boosting generated higher levels of IFN-γ and IL-17A than the DNA boosting.Conclusion: Compared with DNA plasmid booster, immunization using DNA prime/protein boost of pneumococcus protein may be a new strategy to develop vaccines against pneumococcal infection.
The Lassa virus and the Machupo virus belong to the family of the Arenaviridae. They can cause human infection with high mortality and pose a threat to the public health. The arenavirus possesses a cap-snatching mechanism for transcription of viral genome, which is similar to that of other segmented negative-strand RNA viruses. This process involves cleavage of host mRNAs by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase, and the cap-binding by the C-terminal region which has not been verified in Mammarenavirus. To this end, the recombinant expression vectors for the C-terminal region of Lassa virus and Machupo virus polymerase were constructed. The recombinant proteins were expressed in E. coli and was found that the proteins can exist with different oligomerization forms in solution.The oligomerization features by negative staining electron microscopy also were characterized. Finally, initial crystals of the C-terminal of Lassa virus polymerase were obtained, which provides a foundation for further study of its three-dimensional structure and functional mechanism.
Hydroxyl amino acids are a novel kind of amino acid derivatives, which are widely used as a chemically synthetic intermediate.A novel L-leucine-5-hydroxylase (NmLEH) from Nostoc minutum was cloned and inserted into recombinant plasmid, and its expression conditions were optimized. The results showed that when the enzyme was transferred into the BL21 (DE3) host, the induction temperature was 25℃ and the IPTG induction concentration was 0.5mmol/L, the expression levels of NmLEH was highest after induction of 10 (0.45 mg/ml). Using purification process of Ni-affinity chromatography and gel filtration, highly purified recombinant NmLEH was obtained. The enzymatic properties of NmLEH were characterized, and the optimum reaction temperature of the enzyme was 25℃, and the optimum pH was 7.5; the NmLEH enzyme was active at pH 7.0-9.0, and the optimum substrate for the NmLEH were the leucine and methionine. Sequence alignment and phylogenesis analysis implied that residues of H150, H236 and D152 constitute the catalytic triad of NmLEH, which is completely conserved in the Fe(II)/αKG-dependent dioxygenase [Fe(II)/αKG-Dos] superfamily. The formation mechanism of catalytic active site was analyzed based on NmLEH structural model analysis.
Objective: To improve the efficiency of PRV transduction by ethidium bromide (EB) -induced transient demyelination.Methods: 18 adult Wistar rats were randomly divided into muscle group, NS group and EB group(n=6).In the muscle group, 2μl PRV with a titer of 2×10 9 was injected into the tibial anterior muscle and gastrocnemius muscle. NS group was injected with 2μl normal saline (NS) to the sciatic nerve, and 2μl PRV with a titer of 2×10 9 was injected to the same site one week later. In the EB group, 2μl 0.1% EB was injected into the sciatic nerve, and 2μl PRV with a titer of 2×10 9 was injected into the same position one week later. Five days later, perfusion samples were taken and frozen sections were made to observe the infection of neurons at all levels. Methods: A large number of L4-L5 spinal anterior horn neurons, dorsal root ganglion (DRG)neurons, T8 spinal intermediate neurons, C4 spinal intermediate neurons, medulla oblongata, midbrain and cerebral cortex of rats in EB group were labeled by PRV.A small number of neurons at all levels were labeled by PRV in the muscle group and the NS group.Conclusion: EB- induced sciatic nerve demyelination can enhance the reverse transduction efficiency of PRV.
Objective: Programmed death ligand-1 (PD-L1) is an important factor in the immunoregulatory pathway and is one of the important targets in anti-tumor immunotherapy. The PD-L1 knockout mouse model was successfully constructed using CRISPR/Cas9 technology, and its phenotype was initially analyzed.Methods: Cas9 and sgRNA vectors were constructed and transcribed to obtain RNA. RNA was injected into C57BL/6 mouse fertilized eggs by microinjection, and F0 generation positive mice were obtained. F0 generation mice were mated with wild type C57BL/6 mice to obtain F1 generation heterozygous mice, and F2 generation homozygous mouse strains were obtained by self-crossing of F1 generation mice. Subsequently, the expression of PD-L1 gene at the mRNA and protein levels was detected by Real-Time PCR and flow cytometry, respectively.Results: Real-Time PCR and flow cytometry showed that the PD-L1 mRNA expression level and protein expression on PD-L1 homozygous mice with only background signals were significantly decreased, compared with wild-type C57 mice. It was confirmed that the PD-L1 knockout mouse strain was successfully constructed, which provided a new mouse model for PD-L1 gene function research in vivo.
How to deliver bioactive molecules efficiently to target cells and tissues is still one of the challenges for researchers in the field of bioremediation. Until the advent of CPPs, its can mediate a variety of exogenous functional molecules (nucleic acids, polypeptides, proteins, and chemical drugs) into the cell without affecting the function of exogenous active molecules. In addition, CPPs show a more promising advantage in transferring exogenous active ingredients into tumor tissues and cells. Therefore, the classification, identification method, penetrating mechanism of CPPs, and its application in anti-tumor therapy which hope to provide the reference of method and novel strategy for the identification of new CPPs and anti-tumor in clinical were summarized.
Autophagy is a process in which cells degrade intracellular damaged components under stress conditions and involves the involvement of multiple signaling molecules.In the process of disease occurrence and development, autophagy can inhibit or delay the development of the disease, and can also make the disease worse. Therefore, it is of great significance to find the factors that regulate the autophagy at different stages to explore its effective target.Noncoding RNA (ncRNA) is a generic term for a class of RNA that is transcribed from the genome and does not travel to encode proteins. Over the years, more and more different ncRNAs have been discovered and play an important regulatory role in the physiological and pathological processes of animals.Studies have shown that ncRNA plays an important regulatory role in the process of autophagy.This article intends to review the regulation of ncRNA in the autophagy pathway from microRNAs (miRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and to treat diseases such as cancer and Molecular markers provide theoretical guidance and new ideas.
Lysosomal-autophagy system plays a critical part in the adaptive response of cells to nanomaterials. Autophagy has great utility in protecting cells from damage and keeping them stable. However, the essence of autophagy induced by nanomaterials is still unclear. Nanomaterials are recognized as alien invaders, the accumulation of which will activate the body's clearance mechanism. And this will lead to autophagy after the absorption of nanomaterials. This review introduces the self-protection mechanism of autophagy induced by nanomaterials, and analyzes the influence of nanomaterials on lysosomal-autophagy system and their biological effects comprehensively.
Magnetotactic bacteria (MTB) form intracellular membrane-bounded, nano-sized magnetic iron mineral crystals of either magnetite (Fe3O4) or greigite (Fe3S4) called magnetosomes. These magnetic crystals render MTB cells able to navigate in chemically stratified environments by swimming along the Earth’s magnetic field. MTB are morphologically, physiologically and phylogenetically diverse and are widely distributed in aquatic environments. Cells of MTB and their magnetosome nanoparticles have various novel physical and biological properties that can be exploited in a variety of applications. Here we review the current knowledge on the diversity of MTB and summarize the most recent contributions to the field of applications of MTB cells and magnetosome crystals.
Transgenic maize is the second largest transgenic crop in the world and plays an important role in protecting human energy and feed industry. By using the method of patent analysis, this paper makes a statistical research on the patent documents from PatSnap database in the field of genetically modified maize in 1985-2016, the results showed the overall development trend of the global maize patents, the technology distribution and the research hot spot. A comparative analysis of the competitiveness of China’s maize research and development was also carried out. The future of maize industry development was finally put forward.
In recent years, the global development of biosimilars is in full swing. Governments have introduced a series of incentive policies to promote the development and application of biosimilars, in addition, large-scale pharmacy enterprises have also seized biosimilars’ market opportunities through the acquisition of other small and medium-sized biopharmaceutical companies. The Pfizer vs. Johnson lawsuit was taken as a research object, and the overall description of American biosimilars insurance policies, development process of litigation case and in-depth analysis of series focus issues were carried out. The study found that the competitive pressures from the original companies and the clinical conservative attitudes towards the substitutability of biosimilars threaten the application and promotion of biosimilars to a certain extent, while its price advantage and increasingly clear policy environment make the future of biosimilars promising.
Biotechnology and biopharmaceutical industries are the strategic sectors favored by major economies of the world in the 21 st century. Globally, these two sectors have gradually moved into the phase of large scale commercialization industrialization, and are expected to enter a stage of accelerated growth by 2020. They are expected to gradually become a key driver of world’s economy growth. China government has enacted a series of incentive measures and policies to foster a conducive ecosystem in order to accelerate the innovation and growth of the biotechnology and biopharmaceutical industries. With the call for innovation by central government, various provinces and cities of China have built series of biotechnology science parks and incubators to promote the incubation and growth of biotechnology startup companies, in an attempt to move up along the value chain. This paper provides an introduction to the establishment, operation and incubation practice of various successful biotechnology incubators in the United States, it also looks into the collaboration model between these incubators and external parties, from an ecosystem building perspective. Six guidelines were summarized for the successful operation of incubators and three suggestions were given aimed at serving as a reference for those who plan to build or operate a biotechnology / biopharmaceutical incubator.
