Objective: The malignant proliferation and easy invasion and metastasis of breast cancer cells are directly related to their harm to patients. Therefore, it is of great significance to explore the molecular mechanism of breast cancer cells for their effective prevention and treatment. QSOX1 is one of the members of thiol oxidase family. It has been proved that QSOX1 plays an important role in the formation of disulfide bond and extracellular matrix during protein folding. QSOX1 is over expressed in many kinds of cancer cells, including breast cancer and pancreatic cancer. The present study explored the possible role of QSOX1 in breast cancer cell proliferation, invasion and metastasis. Methods: By using CRISPR/Cas9 technology to construct QSOX1 gene knock-out and knock-in models of breast cancer cells, the effects of QSOX1 on the proliferation, invasion and migration of MCF-7 cells were analyzed. Results: The results showed that QSOX1 gene knock-out and knock-in MCF-7 cell lines were successfully constructed by CRISPR/Cas9 gene editing technology. Compared with WT cells, the proliferation ability of QSOX1 KO1 cells decreased significantly, the migration and invasion of cancer cells in vitro were significantly inhibited. However, the proliferation, migration and invasion capabilities of QSOX1 KI cells have been significantly improved. Conclusion: The study initially reveals the role of QSOX1 in the occurrence and development of cancer, and lays an important foundation for further elucidation of its molecular mechanism and design of targeted drugs.
Astrocyte upregulating gene-1 (AEG-1) is one of found in the brain tissue of HIV patients with dementia. In recent years, studies have shown that AEG-1 regulates a variety of central nervous system diseases, but its study on learning and cognition has not been reported. Hippocampus and cortex play an important role in learning and cognition. In this paper, CRISPR/Cas9 technology combined with Cre/loxp system was used to construct hippocampal cortex specific AEG-1 knockout mice, and the correlation between AEG-1 and learning cognition was preliminarily studied on the basis of this model mouse. Firstly, flox homozygous AEG-1fl/fl mice inserted into the loxp site were constructed, and bred with hippocampal cortex specific Cre+/+ recombinase expressing tool mice. Secondly, hippocampal cortex specific AEG-1 knockout mice with AEG-1 fl/fl Cre+ were selected by PCR. Thirdly,Western blot and immunofluorescence were used to detect the knockout efficiency of AEG-1 gene in the hippocampus and cortex. Last but not least, the new object recognition box and 3-chambered social interaction box combined with SMART 3.0 analysis system were used to preliminarily evaluate the learning memory and social interaction behavior of hippocampal cortico-specific AEG-1 knockout mice. Results: The knockout mice with AEG-1 fl/fl Cre+ were successfully obtained.AEG-1 protein expression in hippocampus and cortex of mice was significantly lower than that in control group. The new object recognition results showed that the discriminant coefficient of AEG-1 knockout mice was significantly lower than that of the control group, indicating that the learning and memory ability of AEG-1 knockout mice was weak. However, the social interaction showed that there was no significant difference in social interaction between AEG-1 knockout mice and the control group. These results lay the foundation for future studies on AEG-1 in learning cognition.
Objective: After prokaryotic expression of recombinant antigen CagL (rCagL) from Helicobacter pylori pathogenic island, recombinant antigen rCagL with high purity was obtained by protein purification technology. Anti-CagL polyclonal antibody was prepared and its specificity was also analyzed. Methods:The antigen structure of rCagL was analyzed by bioinformatics software. CagL gene without signal peptide was synthetized by PAS (PCR-based accurate synthesis), and then inserted into expression plasmid pCzn1 to construct recombinant plasmid pCzn1-rCagL. After that, the pCzn1-rCagL plasmids were transferred into E. coli Arctic Express. After expression with IPTG induction, the rCagL protein was purified by Ni-IDA affinity chromatography. The immunoreactivity of rCagL reaction with His label antibodies and anti-H. pylori antibodies was identified by Western blot. Lastly, anti-CagL polyclonal antibodies were prepared by immunizing BALB/c mice with rCagL plus Freund’s adjuvant, and the specificity of anti-CagL polyclonal antibodies was also analyzed by ELISA. Results: Bioinformatics software showed that recombinant antigen rCagL has good antigenic properties. The recombinant plasmid pCzn1-rCagL was identified by double enzyme digestion and gene sequencing, and the nucleotide sequence of rCagL was completely consistent with the theoretical sequence. After induction with IPTG at low temperature of 11℃, the recombinant genetically engineered strains pCzn1-rCagL/Arctic Express can express soluble rCagL protein efficiently by SDS-PAGE analysis. And the soluble rCagL protein accounted for 62.07% of inclusion bodies. The purity of the protein was about 96.6% after purification by Ni-IDA affinity chromatography. Western blot results confirmed that recombinant antigen rCagL could bind specifically to His label antibodies and anti-H. pylori antibodies. ELISA results showed that the prepared anti-CagL polyclonal antibodies could specifically recognize rCagL and H. pylori lysates, indicating anti-CagL polyclonal antibodies had high antibody specificity. Conclusion: The recombinant antigen rCagL can be expressed at low temperature, and the rCagL protein with high purity was obtained after purification. The recombinant antigen rCagL had good antigenicity, and anti-CagL polyclonal antibodies had good immune specificity, which laid a foundation for the development of H. pylori-related diagnostic reagents.
Objective: Using a “double-stranded probe” real-time fluorescent PCR technology to improve the sensitivity of HBV nucleic acid detection, complete the genotype detection of metabolic enzyme CYP2C19 *2 in a tube. Methods: The double-stranded probe and the TaqMan probe was used to simultaneously detect different concentrations of HBV in serum samples by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Then, according to the Ct value of nucleic acid detection by instrument to statistical analysis of results; the double-stranded probe was used to detect samples of different genotypes of metabolic enzyme CYP2C19*2 in a tube, and the detection of nucleic acid Ct value and genotype analysis were performed by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Results: In the detection of HBV serum samples at different concentrations, the fluorescence background of the double-stranded probe was low and the detection sensitivity was higher than the TaqMan probe. And significant differences were noted between the two probes (P<0.05);The metabolic enzyme CYP2C19*2 genotypes of 36 samples were detected using the double-stranded probe, the results were consistent with those of Sanger sequencing. Conclusion: The double-stranded probe real-time fluorescent PCR detection technology can complete the highly sensitive nucleic acid detection of the target gene and also the genotype analysis.
Objective: To explore the efficient assembly technology of virus-like particles (VLPs) of porcine circovirus type 2 (PCV2) and improve the stability of VLPs. Methods: PCV2 Cap protein was expressed in E. coli and self-assembled into VLPs. The stability of VLPs under different ionic strength was analyzed. Disassembly of VLPs was achieved by addition of urea and decreasing pH with tangential flow filtration. Cap protein was obtained by ammonium sulfate precipitation and anion exchange chromatography. Efficient reassembly of VLPs was achieved by removing urea,increasing ionic strength and pH. Results: The stability of self-assembled PCV2 VLPs was poor under 150mmol/L NaCl, and was improved under 500mmol/L NaCl, but it was still easy to aggregate. The nucleic acid content was high. Under the condition of 150mmol/L NaCl, 300mmol/L urea and pH 5.5, VLPs was disassembled. The crude protein was precipitated by 25%-50% saturated ammonium sulfate (V/V) and eluted by anion exchange chromatography under 500mmol/L NaCl to obtain the purified Cap protein with over 95% purity and 65.85% recovery, which the nucleic acid was effectively removed. Urea was removed, the concentration of NaCl was increased to 1mol/L, and the pH was increased to 8.0 with tangential flow technology. The static charge distribution on the protein surface was changed, and efficiently and uniformly reassembly of VLPs was achieved with over 99% assembly efficiency. The stability of VLPs was significantly improved, and was stored stably for more than six months. Conclusion: PCV2 cap protein was obtained by ammonium sulfate fractional precipitation and anion exchange chromatography. Then, the urea was removed, the ionic strength and pH were improved to realize the efficient reassembly of VLPs.
Currently, the 2019 novel coronavirus (COVID-19) caused by SARS-CoV-2 infection is raging around the world and seriously affects human health. SARS-CoV-2 is highly infectious, and the mortality rate of infected severe patients is high. Although various methods are being tried for clinical trials, no authorised effective treatment is available. Mesenchymal stem cells (MSCs) have attracted wide attention in preclinical trials because they have a good therapeutic effect on a variety of human diseases. MSC may use differentiation potential to induce differentiation into functional pulmonary-like cells, immune regulation interacted with various immune cells, inhibit inflammation to reduce the secretion of pro-inflammatory cytokines, migration and homing to damaged lung, antiviral effects through reduce virus replication in lung epithelial cells, and produces extracellular vesicles to repair damaged tissues, thereby gradually recovering the lung function of COVID-19 patients, alleviating and achieving the goal for treating COVID-19. The basic characteristics of COVID-19 and the current main treatment methods of COVID-19 are comprehensively discussed. At the same time, the clinical research and current challenges are analyzed. In addition, the application prospects of MSC for treating COVID-19 are also discussed. In summary, the research provides a theoretical and practical basis for MSC in the treatment of COVID-19.
Coronavirus is a kind of pathogen that can cause respiratory and digestive diseases. At present, the understanding of coronavirus is not deep enough, and the similarities and differences between SARS-CoV-2 and other human coronaviruses in biological characteristics, infection mechanism, epidemiology and clinical characteristics are not clear. By summarizing the characteristics of several coronaviruses including SARS-CoV-2, and analyzes the susceptibility and prognosis of SARS-CoV-2 in special population, so as to provide reference for clinical research and differential diagnosis.
Induced pluripotent stem cells (iPSCs) as well as embryonic stem cells(ESCs),is a kind of cells with potential of self-renewing and unlimited proliferation,has the potentian of inducing differentiation into all types of cells in every germ layer of the body. iPSCs are derived from the body itself, which avoids the immune rejection and medical ethics problems of ESCs and they play an important role in biomedical research. At present, iPS-derived hepatocyte-like cells(iHLCs) have been widely used in the establishment of in vitro and in vivo infection model of HCV.The HCV model based on iHLCs can be used to study the pathogenesis of HCV,the role of host genes in the pathogenesis of HCV, to screen new anti-HCV drugs and development of vaccine.The origin of iPSCs, the research methods of inducing iPSCs into functional liver cells by different strategies, and the application of iPSCs in HCV infection model were summarized.
Type 2 diabetes mellitus (T2DM) is a kind of chronic metabolic disease caused by β-cell damage and insulin tolerance. The rapid growth of its morbidity and high mortality caused by complications has become a medical problem. At present, T2DM is mainly treated with hypoglycemic drugs and insulin sensitizers, but these drugs will have serious side effects. And these drugs can’t control blood glucose and prevent various chronic complications for a long time. Therefore, gene therapy is the main direction of future medical development. Gene therapy can not only target to regulate blood glucose level and improve the effect of lowering blood glucose, but also reduce the complications caused by abnormal glucose metabolism and protect tissues and organs from damage. Based on the understanding of traditional medicine in the treatment of diabetes, the effect and advantages of gene technology in the treatment of T2DM are reviewed, which is not only conducive to the prevention and individualized treatment of T2DM, but also provides a new treatment for diabetic complications.
Mussel foot proteins are a kind of protein complex secreted by mussel foot gland, which have strong adhesion to the surface of substrates due to reactions. Because of its strong adhesion, biodegradability and excellent biocompatibility in the marine environments, it is often used as bio-medical adhesives. However, the extraction of natural proteins is limited by the source of raw materials, and the complex process leads to high price, which hinders the further application and development of mussel foot proteins. The latest development of microbial synthesis provides a new way of thinking for mussel foot protein production and has the significance of expanding the scale of production. The production methods of mussel foot protein by genetic engineering were reviewed, and the applications of recombinant proteins in adhesion antifouling coatings and tissue engineering materials were summarized. At the same time, the research direction is prospected, and it is pointed out that the key technology for the further development of recombinant mussel foot proteins is to analyze the structure activity and hierarchical structure of the protein, on the basis of which the expression level of the protein is improved, so as to obtain more derivatives with biological efficacy.
Mannan oligosaccharides have a series of physiological functions such as, lower blood fat, resist colon inflammation, enhance immunity and regulate intestinal flora and have broad application prospects in the fields of food and medicine. The mannan-oligosaccharide products prepared by the hydrolysis method usually contain monosaccharides, unhydrolyzed polysaccharides, oligosaccharides with different polymerization degree, salt ions and other impurities, which need further separation and purification. The main purification methods of mannose oligosaccharides in recent years are reviewed, including column chromatography, membrane separation, ethanol precipitation and microbial fermentation. The principle, application scope and application examples of various methods are summarized, and the advantages and disadvantages of different methods are analyzed.
As the most common gynecological malignancy, cervical cancer (human papilloma virus, HPV) is the second largest threat to women’s health. Cervical cancer vaccine is an effective way to prevent cervical cancer. Glaxosmithkline (GSK) and Merck (MSD) dominated the global market for cervical cancer vaccines until 2019. Despite the late start of cervical cancer vaccines in China, 20 vaccines have entered the clinical stage driven by domestic innovation policies. In particular, Cecolin, which is developed by INNOVAX and Xiamen University, was approved for market by National Drug Administration in April 2020. It is the first domestically developed cervical cancer vaccine in China and the third in the world. Compared with Europe and the United States and other developed countries, there is still a big gap in the promotion of cervical cancer vaccine in China, the HPV vaccine coverage rate of 9-45 years old women in China is less than 0.05%. In view of the low coverage rate of HPV vaccination, some countermeasures and suggestions were put forward, such as optimizing the review and approval process of HPV vaccine, including HPV vaccine into the national immunization program, and improving women’s understanding of HPV vaccine.
Bevacizumab is one of the most popular subjects for the research and development of biosimilars. Based on the research data of 23 biosimilar sponsors and those of the original manufacturer, combined with the current domestic and international biosimilar guidelines as well as related literatures, the technical assessment key points of the quality similarity of bevacizumab biosimilar were discussed, including the general principles of similarity evaluation of biosimilars, identification of key quality attributes and similarity evaluation of bevacizumab.
