Objective: To establish a colloidal gold technique assay for the rapid detection of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against 2019 novel coronavirus (2019-nCoV) and to evaluate its clinical performance. Methods: The colloidal gold was prepared by trisodium citrate reduction. The receptor binding domain (RBD) of spike protein and nucleocapsid protein (NP) were used as marker antigen. The nitrocellulose membrane was coated with mouse anti human IgM monoclonal antibody and mouse anti human IgG monoclonal antibody, and the detection reagent was prepared by using dinitrophenol-bovine serum albumin (DNP-BSA) and rabbit anti DNP polyclonal antibody as independent quality control. By comparing the clinical coincidence rate of RBD protein and NP protein, the better antigen was selected to prepare the detection reagent, and the performance of cross reactivity, interference reactivity, accelerated stability, specificity and sensitivity of clinical diagnosis were evaluated. Results: The total coincidence rate of RBD protein was 98.48% (389/395), and that of NP protein was 89.11% (352/395). There were no cross reaction with antibody positive samples of 13 common pathogens. Triglyceride, hemoglobin, bilirubin, rheumatoid factor (RF), human anti mouse antibody (HAMA) and antinuclear antibody (ANA) in the samples did not interfere with the test results. The kit was stable after 6 weeks accelerated at 50℃. The sensitivity of IgM was 78.31% (65/83), the specificity was 98.90% (721/729), the sensitivity of IgG was 92.77% (77/83), the specificity was 99.31% (724/729), the sensitivity of IgM and IgG combined detection was 92.77% (77/83), the specificity was 98.35% (717/729), the kappa consistency test had a kappa value of 0.883 0 (P<0.05). Conclusion: The 2019-nCoV IgM/IgG antibody detection reagent (colloidal gold method) has the advantages of high specificity and sensitivity, fast detection speed and portable operation, which can be used as a supplementary method for the existing 2019-nCoV nucleic acid detection method.
Background: Fusarium graminearum is the major pathogen of Fusarium head blight of wheat and other grain crops. To understand mechnism of pathogenesis of the pathogen, molecular analysis has widely carried out during the past dacades. The differential gene expression (DGE) analysis for MGV1 deletion mutant revealed that the MDT1 gene was significantly down-regulated in MGV1 knock-out mutant, suggesting that the gene was involved in the MGV1 pathway. Go annotation given out that the MDT1 gene was involved in the ATP binding and protein dimerization activity. Objective: In order to identify the function of gene, MDT1(MGV1 dependent transcripts)Gene was isolated and characterized. Methods: The split-marker strategy was applied to construct the deletion cassette of the gene and the phenotype and pathogenicity of the deletion mutant were assayed by conventional method. Results: MDT1 gene deletion mutant significantly decreased the amount of conidia and produced less perithecia than that of the wild type, indicating that the gene was essential for the asexual and sexual reproduction. Vegetative growth of the mutant greatly reduced in solid medium. The mutant also was hypersensitive to cell wall degrading enzyme and formed swollen top of the mycelium and fractured hyphea at 32℃in liquid medium, revealing that the gene was related to the cell wall integration in Fusarium graminearum. The complement test confirmed the phenotypic changes of the MDT1 gene deletion. Conclusion: The results demonstrated that MDT1 gene in Fusarium graminearum was involved in conidiation and vegetative growth.
Objectives: To introduce site-directed mutagenesis into the pcDNA3.1(+)-F plasmid containing respiratory syncytial virus F gene coding sequence by single circular PCR using single primer. Methods: First, three single-stranded primers with mutagenesis N70Q, I431N or Q270T were designed according to the template pcDNA3.1(+)-F plasmid, respectively. Second, once-single PCR with the double-stranded DNA template and each of the three single primers was performed. Next, the PCR products were treated with endonuclease Dpn I to eliminate the methylated template DNA and then transformed into E. coli DH5α. Finally, plasmids were extracted from the culture of the selected positive clones and sent for sequencing after digestion identification. Results: The results of enzymatic digestion and sequencing analysis were as expected. Three types of site-directed mutagenesis were successfully introduced, namely ‘_X_’, ‘X_X’, ‘XXX’ when ‘X’ means mutated nucleotide and ‘_’ means the opposite. Conclusions: Single-primer PCR is a simple, quick and effective innovation to introduce site-directed mutagenesis, which solved the problems of multiple PCRs, complicated procedures and low effectiveness in previous methods.
Objectives: Many of biocatalytic redox reactions which are widely used in the production of chiral chemicals involve the regeneration of the coenzyme NADPH in situ. Alcohol dehydrogenases that regenerate NADPH with isopropanol as substrate have the advantages of high specific activity and easy separation of byproduct acetone, attracting more and more attention. Therefore, an alcohol dehydrogenase from Clostridium beijerinckii, namely CbADH, was chosen as the research object for its more considerable specific activity and the most applicable potentiality within present literatures. To solve the problem of poor soluble expression of CbADH in E. coli genetically engineered strains and the consequent enzyme activity as low as 2.31 U / mg DCW, the following studies were carried out. Methods: Firstly, different chaperone proteins were expressed by inducible plasmids to increase the soluble expression level of CbADH, and the results showed that molecular chaperone GroES-GroEL significantly improved the soluble expression of CbADH by 3.57 times more than the original strain, with enzyme activity of 11.18 U/mg DCW which is 4.83 times more than the original strain. Secondly, three other different GroES-GroEL expression strategies were examined: pET-28a(+) single plasmid co-expression, genomic enhancing expression of chaperone, and constitutive-pGro7/pET-28a(+) dual plasmid co-expression. Results: The results indicated that the constitutive-pGro7/pET-28a(+) dual plasmid co-expression strategy had the best effect which improved the soluble expression of CbADH by 8.07 times more than the oringinal strain, with a CbADH activity of 21.79 U/mg DCW, which was 9.43 times higher than the oringinal strain. Conclusions: This study not only lays the foundation for the industrial application of CbADH but also provides a reference for heterologous soluble protein expression.
Organ reconstruction may have severals of requirements for the elasticity, stiffness, and biological activity of the materials due to different applications, but currently many materials are challenging to meet these requirements at the same time. For example, polyethylene glycol diacrylate, a kind of widely used elastic material, do not have high biological activity, while bio-materials such as collagen have poor elasticity. As an elastic functional protein which widely existing in animals, elastin is valued in tissue engineering reconstruction of elastic organs for its special properties that can withstand large deformation without destroying its structure. The amino acid sequence of the elastin-like polypeptide (ELP) designed in this article meets the requirements in the project, excellent biological activity and elasticity, according to the basic repeat unit Val-Pro-Gly-Xaa-Gly and preference and degeneracy of the E coli. condon. After the plasmid was constructed, the ELP was expressed and collected in E coli. BL21 (DE3), and then identified the protein by SDS-PAGE. The modulus data of the protein was tested by rheology, the microstructure of the material system was tested by SEM, the biological activity of the material was tested by cell culture. These methods identified the elastic properties, physical structure and biological activity of materials. They contributed the basis for enhancing the elasticity by crosslinking and its application in tissue engineering and organs reconstruction.
Compared to Cas9, LbCpf1 has higher targeting specificity and other advantages in eukaryotic cells. Therefore, this study aims to obtain the LbCpf1 protein that is cleaved by in vitro activity. To achieve that, pY016 plasmids containing LbCpf1 gene coding region of Lachnospiraceae bacterium ND2006 were double-enzyme digested to obtain the CRISPR-LbCpf1 gene CDS. Next, the prokaryotic expression plasmid CRISPR-LbCpf1-6*His was constructed by ligating the CRISPR-LbCpf1 gene sequence to the prokaryotic expression vector pHis*6(IV) containing the 6*His tag. Afterwards, high yields of recombinant plasmids were obtained from transformed DH5α competent cells. Then the obtained plasmids were identified by double-enzyme digestion and sequencing, the results of which showed the correct recombinant plasmids were constructed successfully. The correct plasmids were subsequently transformed into E. coli BL21 (DE3) competent cells to generate a BL21(DE3) expression strain containing the recombinant plasmid CRISPR-LbCpf1-6*His, which were then inoculated and cultivated at 37°C, on a 160 r/min shaker. The expression of target gene was induced by IPTG (final concentration 0.5 mmol/L) for 5 hours, and the production was purified by Ni column affinity chromatography, dialysis and desalting, SDS-PAGE gel electrophoresis and other steps to obtain the recombinant protein. The final concentration of the protein can reach approximately 400 ng/μl. Finally, via in vitro cleavage assay, it showed the protein was able to process the pre-crRNA in an appropriate environment and bind to the mature CRISPR RNA (crRNA) to cleave the target DNA in vitro, which verified the recombinant protein cleavage activity. In conclusion, this study provides a method to obtain high-purity LbCpf1 protein, supporting the usage of LbCpf1 in further genetic editing research.
Pasteurella multocida (Pm) is an important pathogen that causes respiratory diseases in pigs, which has brought huge economic losses to the world pig industry.Accurate, sensitive and rapid Pm detection method is helpful to understand the prevalence of Pm in clinical practice, so as to take corresponding prevention, treatment and comprehensive prevention and control measures.In this paper, the status, principles, advantages and disadvantages of the research on the etiology, molecular biology, immunology and molecular typing methods of Pm are reviewed in order to provide references for the further establishment of the standard detection methods of Pm.
Different factors affect DNA integrity, which can be divided into endogenous and exogenous factors, these factors can cause different degrees of DNA damage. Among them, DNA double-strand break is the most serious kind of DNA damage, which is characterized by two DNA strands being cut off. If it is not repaired in time, it will cause a series of damage reactions. Serious DNA double strand break can even cause severe consequences such as cell apoptosis and tumor. Therefore, rapid and accurate detection of the degree of DNA double-strand break in cells is helpful to assess DNA integrity, the effects of genetic toxicity in and out of the environment, clinical diagnosis and chemoradiotherapy monitoring. DNA double-strand break detection technology has developed rapidly in recent years. According to the principle, it can be roughly divided into physical or chemical methods, immunofluorescence method and high-throughput sequencing method. These methods detect the physical and chemical characteristics of the products after DNA double-strand break or specific molecular markers, so as to evaluate the degree of DNA double-strand break. The latest research progress, application and advantages and disadvantages of these methods were introduced in order to provide guidance for the future research and clinic of DNA double-strand break detection.
Recently, continuous cell culture is becoming the process focus in the pharmaceutical industry due to its high volumetric productivity, stable product quality attributes, and cost- effectiveness. Compared to the traditional fed-batch culture, benchtop-scale perfusion culture requires quantities of media and labor costs due to its longer culture duration and operation complexity, thus failing to satisfy the current requirement of accelerated and efficient process development. To obtain a robust perfusion process with reduced costs, high-throughput perfusion models are utilized for batches of small-scale perfusion culture in the early-stage process development including clone screening, media selection and process parameter optimization, providing practical process data for late-stage large-scale bioprocessing. Furthermore, they are also applied to predict the phenotype and product quality attributes in large-scale culture. This article will focus on the characteristics, applications and comparisons of current high-throughput systems including shake flasks and spin tubes, parallelized automated ambr systems and microfluidic systems, and discuss the opportunities and challenges faced with high-throughput perfusion models in the bioprocessing development, then look forward to the future prospects.
Monoclonal antibodies and antibody fragments play important roles in the pharmaceutical market. They are mainly produced in mammalian cell systems, which have several limitations such as complex manipulation and high costs. For purpose of using cheap drugs, monoclonal antibodies and antibody fragments have been produced in prokaryotes and yeasts. However, the lack of glycosylation and the low yield of antibodies prevent their development. The progress has been made recently in the prokaryotes and yeasts to enhance the antibodies production via optimization of transcription and translation, co-expressing chaperones and inhibiting proteolytic degradation, etc. This will lay the foundation for the industrialization production of antibodies in prokaryotes and yeasts.
The cytochrome P450 oxidase can catalyze a series of regiospecific and stereospecific chemical steps and participate in the synthesis of many natural products such as terpenoids, sterols and alkaloids. Terpenoids are a large class of compounds in active natural products and have important value in the fields of medicine and perfume. Terpenoids require P450s for biosynthesis and post-modification, but at present, the lower catalytic activity of known P450s greatly limits the efficiency of terpenoid biosynthesis. Therefore, it’s urgent to discover and modify highly active P450s for terpenoid biosynthesis to fully realize its immense industrial application potentiality. This article reviews the different P450s families in terpene metabolism and recent advances in the discovery and engineering of P450s in terpene biosynthesis. Furthermore, it highlights the main strategies of synthetic biology in broadening the application of P450s in terpene synthesis. Feasible strategies have been proposed to further accelerate the discovery of P450s and P450s engineering, based on synthetic biology technology, this article put forward suggestions and prospects for the future application of P450s in terpenoid synthesis.
