Objective:To generate the human bone-seeking breast cancer cell line expressing Cas9 protein and study its biological and ultrastructural properties for the research of breast cancer cells' bone Metastasis using CRISPR-Cas9 technology. Methods:Cas9 protein gene was transfected into the human bone-seeking breast cancer cell line MDA-MB-231BO by lentiviral vector. After G418 screening, MDA-MB-231BO-Cas9 cell line which can constantly express Cas9 protein was obtained and examined by flow cytometry, Western blot and immunocytochemistry; Cell proliferation and migration were verified by real time cellular analysis and wound-healing assay; the effect of Cas9 expression on the ultrastructure of cells was observed under the transmission electron microscope. Results:The human bone-seeking breast cancer cell line MDA-MB-231BO-Cas9 expressing Cas9 protein has been constructed successfully, and the expression of Cas9 has no effect on cell's proliferation, migration and ultrastructure. Conclusion:MDA-MB-231BO-Cas9 cell line can be used for the further research using the CRISPR/Cas9 technology.
Interferon α2b (IFNα2b) is a multifunctional cytokine for the treatment of viral diseases and tumor diseases, and its clinical applications are limited by the short half-life. IFNα2b was fused to the C-terminus of human serum albumin (HSA) and the pMH3 plasmid containing fusion protein (HSA-IFNα2b) encoding gene was constructed. Fusion protein expression Chinese hamster ovary (CHO) cell line was established by electroporation and high expression cell lines were screened by adding G418. The expressed fusion protein was analyzed by Western blot and results showed that the expressed protein was detected by both anti-bodies of HSA and IFNα2b. In suspension culture, fusion protein concentration achieved 65mg/L at the end of batch fermentation. Different generations of fusion expression cell lines showed the similar cell growth and protein production and thus a stable expression cell line was obtained. In 3-L fed-batch culture, the optimized culture time was 15 days and final HSA-IFNα2b production was 121mg/L. After two-step purification, the purity of fusion protein was 96.8% and the total yield was 22.3%. According to the "Chinese Pharmacopoeia 2015 edition" of the IFNα2b detection method, the results show that the anti-virus activity is 4.1x106IU/mg. In conclusion, fusion protein of HSA-IFNα2b was first expressed in mammalian cell CHO and the obtained fusion had high biological activity.
Purpose:To explore whether soluble corin can be activated by PCSK6 (the proprotein convertase subtilisin/kextin type 6) and exhibited biologic activity. Methods:To construct soluble corin expression vector, corin cDNA fragment (463~3219bp) was amplified using pcDNACorin as a template, and cloned into the eukaryotic expression vector pSec/FRT/V5-His-TOPO to yield the plasmid pSECsolCorin. The constructor was verified by restriction endonuclease digestion and DNA sequencing. Then soluble corin, PCSK6 and pro-ANP expression vectors were transfected into HEK293 cells, collected cell lysate and conditioned medium. Western blot were used to determine protein expression. Then soluble corin medium was incubated with increasing amounts of PCSK6 for 1h, activated corin and PCSK6 were detected by Western blot. Then also incubated activated soluble corin with pro-ANP to exam corin activities. Results:Construct soluble corin expression vector, and soluble corin can be expressed in HEK293 cells. When soluble corin was treated with condition medium contain increasing amount PCSK6, activated corin protease fragments (corin-p) can be detected and increased in dose-dependently manner. And activated soluble corin can process pro-ANP to ANP in dose-dependently manner. Conclusions:Soluble corin can be activated by PCSK6, then exhibiting the capacity of processing pro-ANP. Soluble corin may become a new therapeutic strategy of heart failure.
Immunoassay, based on antigen-antibody reaction, was one of the common methods employed to detect AFB1. In order to prepare antibody against AFB1, A codon-optimized DNA sequence (suitable for expression in Escherichia coli (E. coli)) of high homology was synthesized according to comprehensive screening of a large number of anti-AFB1 VHH sequences obtained through phage display library. A phage antibody library was constructed by introducing random mutations in part of CDR2 and CDR3 regions of the synthesized anti-AFB1 VHH. Anti-AFB1 VHH of high affinity was screened through phage-ELISA, using AFB1-OVA as coating antigen. After four rounds of screening, 15 clones capable of binding to AFB1 were obtained. VHH gene of the clone with highest binding ability was amplified, and applied to construct expression plasmid pET-22b-VHH. The corresponding VHH protein was expressed in E. coli BL21(DE3). According to indirect competitive ELISA, the sensitivity of anti-AFB1 VHH obtained was about 10μg/ml.
14-3-3 proteins are a family of conserved proteins that interact with numerous partner proteins in a phospho-specific manner, and can affect the target proteins in a variety of ways. prokaryotic expression vector of 14-3-3 gene was constructed, and the recombinant 14-3-3 protein was purified, then the 14-3-3 antibody was obtained using this recombinant protein. To characterize the aluminum (Al) tolerance of 14-3-3 protein in yeast, a transgenic Saccharomyces cerevisiae containing pYES3/CT-14-3-3 was generated. The results of growth showed that the transgenic yeast grew better than the control strain on plates with 5mmol/L Al. These results suggest that 14-3-3 conferred Al tolerance in yeast by promoting the growth of yeast.
Human enterovirus 71 (EV71) is one of the main causative pathogens for hand-foot-and-mouth disease (HFMD) which often infects infants and children under 5 years old. Although prophylactic vaccines of inactivated EV71 virus for HFMD have been clinically available, low productivity and safety concerns still prompt scientists to develop other vaccine substitutes. Recently, a kind of vaccine candidate VacA for EV71 has been designed and reported, which contained the main antigen peptides, was fused as a single protein and expressed through recombinant E. coli system. This candidate was demonstrated to have high neutralizing antibody titers as well as efficiently protect against virus infection. However, forming inclusion bodies in E. coli and failure of being separated from the host contaminants through route chromatographs hampered its path to go forward to whole pre-clinic and safety assessments.Another form of VacA as His-VacA was constructed by inserting a His tag at the N-terminal of VacA and explored to be separated through metal affinity chromatography. As still forming inclusion bodies, the fusion protein of His-VacA was directly purified at denatured state through a special mental affinity medium of cOmplete His-tag, which could tolerate low concentration of EDTA and DTT. Purity of about 95% for His-VacA was achieved after such one step chromatographic procedure, with a recovery of 46.8%. Dialysis was then adopted to remove urea in the purified protein, but protein almost completely precipitated when dialyzing against PB buffer without any additives. Nevertheless, in the case that the purified denatured protein was firstly diluted in a buffer containing 2mol/L urea and then exchanged to a buffer without urea through a desalting column of G25, no protein precipitate could be found, resulting in nearly 100% recovery. More inspirationally, His-VacA was found in the form of virus-like particles with a size of 10nm through transmission electron microscopy. Moreover, this protein particle is very stable in the final buffer and then could be used for the further pre-clinic assessments. All these results lay a foundation for developing His-VacA as a new kind of EV71 vaccine with the advantages of safer and cheaper.
To establish CRISPR/Cas9-edited FGF5 cell strains, we designed gRNA targeted sites around the first extra of the FGF5 gene and constructed two vectors by Cas/gRNA plasmid construction kit weredesigned. vectors into cashmere goat fibroblasts by electroporation respectively was transfected. T7 endonuclease 1 (T7E1) was used for the detection of mutation efficiency. The best vector was transfected into cashmere goat fetal fibroblasts and all the monoclones was cultured. Then all the cell colonies by sequencing were idenfified. Sequencing results demonstrated that CRISPR/Cas9 was available for FGF5 gene edited and 20 FGF5+/- and FGF5-/- cell colonies were obtained, and the effiency was 14.81%. The double mutated cell colonies could be used as donor cells to construct reconstructed embryos which provide the possibility in production of FGF5 edited cashmere goat in the future.
As a common DNA polymerase, Bst DNA polymerase large fragment has been a kind of important DNA multiple displacement amplification enzyme, which can cause chain reaction, high fidelity, high temperature resistance and so on. Objective:Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. In order to reduce cost, a high yield, convenient and high activity of Bst DNA polymerase large fragment expression system had been designed, and the conditions in the amplification of the gastric cancer genomic DNA from FFPE tissues were explored. Methods:Bst DNA polymerase large fragment was cloned and expressed by the PTWIN1 plasmid, and purified by the chintin column. It was used to amplify the Human genome DNA at different temperature to explore the optimum temperature. Then the gastric cancer genomic DNA from FFPE tissues was amplified on the optimum condition. Results:The Bst DNA polymerase large fragment yielded a median 200-fold of DNA amplification. Conclusion:Bst DNA polymerase large fragment is suitable for WGA of DNA extracted from FFPE tissues. Amplified DNA may be used for the detection of gene copy number changes and genome profiling by array CGH.
Objective:To screen the protein related to antibiotics biosynthesis in Micromonospora carbonacea JXNU-1 with broad-spectrum antimicrobial activity. Methods:iTRAQ was used to analyze the differentially expressed proteins between the time of antibiotics secretion/non-secretion in M.carbonacea JXNU-1. Results:A total of 2 390 proteins were identified using iTRAQ, and 172 proteins were differentially expressed during the antibiotics secretion time in M.carbonacea JXNU-1, including 76 up-regulated and 96 down-regulated. Twelve proteins and five gene clusters closely related to biosynthesis of antibiotics were screened based on the functional analysis of the differentially expressed proteins with GO and COG, annotation. Conclusion:The proteins related to antibiotics biosynthesis in M.carbonacea JXNU-1 have been screened based on iTRAQ, it could provide the theoretical basis for revealing the mechanism of antibiotics biosynthesis in M.carbonacea JXNU-1.
Gluconobacter suboxydans is an important industrial microbiology and can incompletely and efficiently oxidize a great variety of carbohydrates, alcohols and other polyols to form the corresponding aldehydes, ketones and organic acids and other products. The use of homologous recombination technology to transform genomic modification is an effective means of industrial breeding. There are many defects when use antibiotic marker as the screening marker in traditional methods. The construction of uracil phosphoribosyl transferase gene deletion strain is to achieve Gluconobacter suboxydans seamless genome editing. The suicide plasmid pMD18-Jqupp is transformed into wild type Gluconobacter suboxydans J12 and the mutant which knocked encoding uracil phosphoribosyl transferase gene was screened by tetracycline resistance and 5-fluorouracil. The physiological validation results show that the mutant strain can grow on medium containing 0.5mg/ml 5-fluorouracil, but the mutant covering upp gene can't grow on medium containing 0.5mg/ml 5-fluorouracil. It proves that the upp gene can be used as a negative selection marker in G.suboxydans-upp mutants to achieve Gluconobacter suboxydans genome modification and transformation by twice homologous recombination. It provides the foundation of changing Gluconobacter suboxydans to obtain valuable industrial strains using metabolic engineering in the future.
Oxytetracycline (OTC) is a broad-spectrum antibiotic produced by Streptomyces rimosus. OtcR was confirmed to be the pathway-specific activator of OTC biosynthesis to directly activate the oxy (oxytetracycline) cluster. OTC production was significantly improved by overexpression of OtcR under the strong promoters. Overexpression of OtcR increased OTC production dramatically by 4 times compared to the parental strain S. rimosus M4018. For a further improvement of the OTC production, the intracellular pool of malonyl-CoA by overexpressing acetyl-CoA carboxylase in M4018 were increased. Herein, for the OTC production, that by overexpressing both the pathway-specific activator OtcR and acetyl-CoA carboxylase in S. rimosus had been shown, a maximum of 9.09g/L was achieved in recombination strains, while only 1.37g/L in the wild type strain M4018.The work has an important significance for engineering industrial strains to improve OTC production.
Saccharomyces cerevisia is generally regarded as safe and widely used in industrial fields. Fine-tuning the expression level of key genes involved inβ-carotene biosynthetic pathway is the key issue for β-carotene production in S. cerevisia. β-carotene producing strain library was constructed by integrating heterologous genes (crtE, crtI, crtYB) from Xanthophyllomyces dendrorhous through Delta site integration.28 strains in darker yellow were picked out and the titer was testified in 96 well plate coupled with shake flask. There were significant difference inβ-carotene titers, ranging from 5.70mg/L to 61.88mg/L. Then the strain with highest titer(SyBE_Sc118012)was selected for further engineering. By means of integrate one copy of tHMG1 and fusion gene BTS1-ERG20 at endogenous ypl062w gene site, the production ofβ-carotene was improved by 1.65 fold, up to 162.1mg/L, which is the highest reported titer at shake flask level. The research highlighted the importance of Delta site integration together with precursor enhancement strategy in heterologous biosynthesis in S. cerevisia.
CRISPR/Cas system widely exists in the bacteria and archaea which is an adaptive immune mechanism of protection by mediating the degradation of exogenous DNA to achieve resistance against invading viruses and exogenous DNA and it is a newly developed genome fixed-point editing techniques. The basic structure, mechanism of action, classification, application and other aspects of the CRISPR/Cas system were introduced, and analyzes the prospect of applying it in animal genetic improvement.
Therapeutic antibody drugs with specificity and efficiency have been developed for different indications.The therapeutic antibody drugs have frameworks from IgG, most of which belong to the IgG1 isotype. Due to various subclasses with different structure and function, affect the physical and chemical properties, biological activity and the ability to trigger effector function and so on, in the process of the research and development of therapeutic antibody drugs, it is important to choose the suitable antibody subclass in design to achieve the desired effect and reduce side effects. The curative related effect of choosing IgG subclass and the development of selecting of IgG subclass for the research and application were reviewed, in order to provide new ideas for therapeutic antibody drugs development.
Mortierella alpina is a significant oil-producing fungi in which oil content reached up to 50% of dry cell weight. It contains a wide range of polyunsaturated fatty acids and its content is higher than other strains. Recently, molecular biology research for Mortierella alpina genetic modification system has made great progress. The establishment of Mortierella alpina genetic modification system is important to improve the level of polyunsaturated fatty acids. The latest application of genetic modification system for Mortierella alpina was reviewed, including transformation methods, types of selective markers, their advantages and disadvantages, and the application of the transformation system.
Objective:To analysis status and trends of synthetic biology in the perspective of product developments. Methods:Based on the Synthetic Biology Products and Applications Inventoryunveiledby Woodrow Wilson International Center for Scholars' Synthetic Biology Project,supplementary search and analysison development phase, potential applications and prospect for the future were performed. Results:To 2015, at least 81 companies (or research institutions),mainly US, with 116synthetic biology products have been developed. The products mainly concentrated in chemicals and medicines. Conclusion:Synthetic biology has created a paradigm shift whereby the analysis of biology is being supplanted by its synthesis, and their product developments have the potential to significantly impact approaches across a variety of industries.
The quality of cell drug preparation is directly related to the curative efficacy of cell therapy. Due to the reason that the cells for cell therapy have biological effects, the preparation technology and application solution for cell drug are of diversity, complexity and particularity, unlike the traditional biological drugs that have a unified production standard. The preparation process of cell drugs mainly include donor screening, donor detection, collection, processing, separation and purification, storage and so on. The preparation technologies of several cell drugs including hematopoietic stem cell, mesenchymal stem cell, hepatic cell and dendritic cell are briefly introduced.
