The aim is to construct the stable mammalian cell lines which express the Aβ-specific single-chain fragment variants (scFv). In order to construct the scFv gene, the variable regions of light and heavy chain from Aβ-specific monoclonal antibody (A8) were used as the template, and splicing overlap extension polymerase chain reaction (SOE-PCR) was used to get the scFv DNA fragments. Short sequence (G4S)3 or p2A was selected as the linker for scFv construction. The eukaryotic expression vectors for anti-Aβ scFv was constructed. Hela and CHO cells were transfected via Lipofectamine 2000, and the scFv products were detected through Western blot. The stable cell lines were screened with hygromycin B, and the antigen-binding ability of scFv in supernatants was detected by indirect ELISA and dot blot. The cellular protection ability of scFv was identified by cell transmission electron microscopy (TEM). Three eukaryotic expression vectors, pSecTag2/HygroA-VL-(G4S)3-VH, pSecTag2/HygroA-VH-(G4S)3-VL and pSecTag2/HygroA-VL-p2A-VH were constructed; and two cell lines expressing the Aβ-specific scFv, Hela-VL-p2A-VH and CHO-VL-(G4S)3-VH were established. The results of indirect ELISA and dot blot showed that the supernatants could recognize Aβ specifically, and the results of cell assays in vitro showed that the supernatants could block the cytotoxicicity induced by Aβ oligomers. The stable cell lines expressing anti-Aβ scFv could be used for further AD immunotherapeutic study.
Objective: To investigate the effects of S100A6 on cell proliferation and migration of cervical cancer cells and explore its mechanism. Methods: Quantitative polymerase chain reaction (qPCR) was used to detect the basic expression of S100A6 in HeLa, SiHa and CaSki cells; HeLa and SiHa cells were treated with recombinant adenoviruses AdS100A6 and AdsiS100A6 to upregulate and downregulate S100A6 expression respectively, Western blot was used to verify the effects of virus infection. MTT method was used to detect cell proliferation ability; wound healing assay was used to detect cell migration ability; Western blot was used to detect the level of E-cadherin, N-cadherin and p-Akt; reverse transcription and polymerase chain reaction(RT-PCR) was used to detect the level of Snail and Twist, the downstream target genes of PI3K-Akt pathway. Results: In AdS100A6 group (HeLa cells), the OD492 value and wound healing rate at the 3rd day were increased, N-cadherin was increased, but E-cadherin was decreased; In AdsiS100A6 group (SiHa cells), the OD492 value at the 5th day and wound healing rate at the 3rd day were decreased, N-cadherin was decreased, but E-cadherin was increased; At the same time, in AdS100A6 group, p-Akt was increased, Snail and Twist were also increased obviously. Conclusion: S100A6 promotes cervical cancer cells proliferation and migration and the effect may be mediated by inducing epithelial-mesenchymal transition and activating PI3K/Akt signaling pathway.
Objective: To study the effect of tripartite motif 5 alpha (TRIM5α) on the HIV-1 infection of CD4+ T cells of Macaca mulatta. Methods: Separation CD4+ T cells from peripheral blood of Macaca mulatta by magnetic cell sorting and the rate of CD4+ T cells was detected by flow cytometry. The transcription activator-like effector nuclease (TALEN) plasmids which targeted TRIM5α gene were constructed and transfected into CD4+ T cells by electroporation. The cells which transfected into TALEN plasmids were separated by flow cytometry. The genome of the targeted cells was extracted and analyzed by T7E1 enzyme digestion. The HIV-1 virus were used to infect the CD4+ T cells which were targeted TRIM5α and the ability of virus infection was detected by ELISA. Results: The CD4+ T cells were successfully separated from the peripheral blood of Macaca mulatta and the positive rate was 99.5%. The TRIM5α TALEN plasmids could enter the CD4+ T cells by electroporation, the infection efficiency was 24.8% and the target efficiency was 40%. The result of ELISA showed that the HIV-1 virus could infect the CD4+ T cells of Macaca mulatta which were targeted TRIM5α. Conclusion: Targeting TRIM5α gene of CD4+ T cells of Macaca mulatta can increase its ability to infect HIV-1 virus. This will lay the foundation for the further establish the Macaca mulatta model for HIV-1 study.
Objective: The object is to construct prokaryotic expression vector which carried mouse cloned c-Myc gene, and achieve its expression and then purify the product.Methods: TetO-FUW-OSKM plasmid was used as a templet to amplify c-Myc gene fragment by polymerase chain polymerization reaction (PCR). After inserting into pET3C vector, the c-Myc gene was transformed into E.coli DH5α. The eligible recombinant plasmid pET3C-Myc was screened out after resistance selection, restrictive identification by PCR, and sequencing and then transformed into E.coli Rosetta (DE3).Result: The result of DNA agarose gel electrophoresis show the amplified gene met the size of destination gene. The sequencing of the reconstructed gene was the same as that of in GenBank. Reaching the destination, the purified product proved to have about 64kDa by SDS-PAGE test. Conclusion: It is successful to construct the recombinant plasmid pET3C-Myc, and achieve its expression in the Rosetta (DE3) and purify the product. The above success paves the path for next study.
To obtain high-efficiency soluble expressed apoptin gene of chicken anemia virus, the apoptin gene sequence was optimized based on E. coli codon preference and inserted into pBCX plasmid vector harbored msyB chaperonin, and then transformed into the competent into E. coli BL21 (DE3) to induce expression, followed by identification of SDS-PAGE and purified by nickel column affinity chromatography. The activity of anti-tumor was evaluated using microscopic examination, CCK 8 experimental determination and DNA ladders tests. The results showed that the E. coli expression vector pBCX-CAV-Apoptin was successful constructed. A large amount of soluble expression product was obtained in E. coli under the normal cultuered condition at 37℃. The fusion protein weighted 42 kDa, and 1.5 mg of purified recombinant proteins could be obtained from 50 ml bacteria liquid. CCK 8 experiment results showed that the purified recombinant protein could induce the growth inhibition rate over 60% of lymphoma JEKO-1 and REC-1 cells post treated 36 h. Microscope and DNA ladders tests intimated that the recombinant protein could induced the apoptosis of JEKO-1 and REC-1 cells rather than HUVEC cells. Western blotting tests indicated that the recombinant protein activated the expression of caspase-3, but not the cleavage of caspase-8 in JEKO-1 cells. It showed that the fusion protein msyB-CAV-apoptin could achieve efficient soluble expression, and the expressed products had specific antitumor activity.
A 2 064 bp upstream sequence of translation initiation site of OsHAK26, a member of KT/HAK/KUP potassium transporters gene family in rice was analyzed and that included a number of elements in response to the development, hormones, and abiotic stresses and common promoter elements of KT/HAK/KUP family, in addition to the basic promoter elements such as TATA-Box, CAAT-Box and so on. Subsequently, the fragment together with other four different 5'-end deletion fragments (－1 473bp、－963bp、－441bp、－193bp) of the OsHAK26 gene 5'-flanking sequences were inserted into the plant transient expression vector pBI221 to replace CaMV 35S promoter respectively and the transient expression analysis was conducted in Arabidopsis mesophyll protoplast system. The result showed that the five fragments had promotor activity decreasing with the length decrease. but deletion of －963bp~－441bp had led to a significant rebound in activity, so it inferred that the section contained suppression components.－441bp~+95bp region activity suddenly surged,so the －441bp~－193bp was core region of promoter of OsHAK26.
Protein synthesis is a complex dynamic process, its abundance is a final measurement of gene expression level. Functional important proteins are always highly expressed in most of tissues. The protein abundance of A. thaliana from PaxDB database and computed translation elongation index (ITE and CAI) of protein coding genes of A. thaliana by both DAMBE and codon W were compiled, and the correlation between protein abundance and translation elongation efficiency was analysed,especially used logarithm in the analysis. The results showed that ITE is better than orignal CAI to analysis, high expressed genes have similar expression level in different tissues in A. thaliana and there was clear correlation between protein abundance and ITE in A. thaliana.
Objective: AuMan5Aloop, a hybrid β-mannanase, was constructed by substituting the loop structure of wild-type AuMan5A with the corresponding one of Aspergillus fumigatus β-mannanase. To analyze the correlation between the enzymatic properties and amino acid H321 of hybrid β-mannanase, its H321 was mutated into the corresponding Gly of AuMan5A. Methods: Using the mega primer PCR method, the mutant enzyme gene, Auman5Aloop/H321G, was constructed by mutating a H321-encoding codon CAC of Auman5Aloop into a Gly-encoding GGT. Then, Auman5Aloop/H321G was expressed in Pichia pastoris GS115 mediated by expression plasmid pPICZαA, and the enzymatic properties of expressed recombinant enzyme, AuMan5Aloop/H321G, were analyzed. Results: Before and after the H321G substitution, the temperature optimum (Topt) of AuMan5Aloop or AuMan5Aloop/H321G was 75℃, higher than that (65℃) of AuMan5A. The half-life at 70℃ (t1/270) of AuMan5Aloop/H321G was 300 min, between AuMan5A (10 min) and AuMan5Aloop (480 min). Besides, its specific activity was 12.8 and 1.43 fold those of AuMan5A and AuMan5Aloop, and its catalytic efficiency (kcat/Km) was 14.1 and 1.12 fold those of the latter two, respectively. Conclusion: After H321G substitution as well as determination and comparison of temperature characteristics, specific activity and catalytic efficiency of three enzymes, the certain effect of H321 on the enzymatic properties of AuMan5Aloop was confirmed.
To screen strains which could selectively producing elevated molecular weight and pigment-free pullulan for the production of pullulan industry, a new strain was separated and then the culture condition was optimized. The improved YPD solid culture medium (addition of 1mg/L chloramphenicol and 0.5 g/L Trypen Blue) was used as selection medium. A new culture, strain A5, was screened and identified based on the morphology, colony and ITS interval sequence analysis. Single factor test was performed to optimize the fermentation conditions. The separation and purified production was identified and determined with Thin-layer chromatography (TLC), Fourier-transform infrared (FT-IR) and Gel permeation chromatography (GPC). The new strain was identified to be Aureobasidium pullulans, and named as A. pullulans A5, which could selectively produce pullulan. The fermentation conditions were optimized to be 8% (w/v) maltose, 1% (w/v) yeast extract, 2% (w/v) peptone, 0.5% (w/v) K2HPO4, 0.06% (w/v) (NH4)2SO4, 0.03% (w/v) CaCl2, initial pH6, 7% (v/v) inoculation amount. TLC and FT-IR spectroscopy of this production were consistent with those of the standard, and the molecular weight was 63.84 kDa. A yeast-like fungal strain A5 was successfully obtained and identified with its high molecular weight and pigment-free production. At the optimized conditions, the pullulan yield was 22.9 g/L. So a candidate strain for the production of pullulan industry was provided.
It has quite significance and broad application prospects to develop novel strategies to produce special functional fatty acids with high added value by oleaginous microorganisms. Two kinds of yeasts S. cerevisiae and Y. lipolytica were selected to test how to selective uptake and increased accumulation of free saturated fatty acids. The results shown that when C ≤ 10 carbon chain length,free saturated fatty acids couldn't be uptaked and utilized on the contrary to suppresses the growth of cell, especially C≤ 8 cells were killed quickly; When C = 11, it had certain inhibitory effect on the growth of cells which leads to the weak growth of bacteria; When C ≥12, it had no impact on cell growth and cells grew well; Even C fatty acids was digested faster than the base C fatty acids; Lipid body could be visualized by fluorescence microscopy of cells stained with Nile red. No significant variations were observed in S. cerevisiae or its mutants grown on YNBD. Where only a few small LB were observed in Y. lipolytica grown on YNBD. After growth YNBF medium, Y. lipolytica strain contained several larger LB and a few smaller ones in cell; The experiment of GC/Mass analysis indicated that the most of fatty acids except long-chain fatty acid (C≥16) could be uptaked and accumulated by S. cerevisiae S228C BY4741-pox1 or S. cerevisiae S228C BY4741-pox1, 3 from culture medium, however medium-chain fatty acid couldn't be accumulated by original S. cerevisiae S228C BY4741. Thus, yeast could uptake and accumulate saturated fatty acid based on different carbon chain length. The most of fatty acids except long-chain fatty acid (C≥16) could be uptaked and accumulated by S. cerevisiae S228C BY4741-pox1 or S. cerevisiae S228C BY4741-pox1, 3 mutants from culture medium. The most of fatty acids also could be uptaked by Y. lipolytica from culture medium in cell.
Single-cell PCR uses the DNA or RNA contained in a single cell as template for the amplification.The quality of the template is one of the key factors for the success of the reaction.Three different proteinase K cell lysate buffers (SDS cell lysate buffer,NP-40 cell lysate buffer and Tween-20 cell lysate buffer) were used to digest the matured oocytes of porcine in vitro.The product of digestion was used as the template for single-cell PCR amplification directly.The results showed that the quality of template processed by NP-40 cell lysates buffer was the best,with a high amplification efficiency of 95%.The amplification efficiency of PCR with template produced by Tween-20 cell lysates buffer was about 65%;while there was no positive bands were detected using the SDS cell lysates Buffer.Combined the single-cell PCR with the microinjection techniques,and it has been used to detect the activity of CRISPR-Cas9 target in vitro.The results showed that this detection method has a high efficiency and stability.
The target genes of cystathionine β-synthase (CBS) and cystathionine β-lyase (CBL) from Saccharomyces cerevisiae were amplified by PCR and cloned into the expression vector pET28a respectively. The resulting plasmids were then transformed to E.coli BL21(DE3) cells. After induction of the gene expression with IPTG and protein purification, the purities of recombinant CBS and CBL were both near 90% with a final yield of 80%. The soluble expression levels of CBS and CBL were 26 mg/L and 332 mg/L respectively, and the specific activities of purified enzymes were determined to be 15 U/mg and 72 U/mg. With utilization of recombinant CBS and CBL as two key components, an enzymatic cycling homocysteine (Hcy) assay kit was developed and characterized preliminarily, whose functionality and stability are believed to meet the requirements of in vitro diagnostic usage. In addition, functionality comparison of our Hcy assay kit with the imported product that has been sold on the market did not show any obvious difference.
The conventional treatment of spinal cord injury (SCI) is decompressive laminectomy within a given time after SCI to prevent the secondary damage. Theoretically, cell replacement therapy can cure SCI, different types of cells can exert therapeutic effects from various aspects including injured spinal cord axon regeneration, neuron reconstruction and remyelinization, and finally promote the functional recovery. The published literatures in recent years which focusing on stem cell therapy in spinal cord injury were reviewed.
Currently, there are enormous challenges for the research on differentiating complex traits in the field of biogenetics, and many methods are employed to tackle these challenges, among which molecular marker, QTL mapping and sequence analysis are useful in targeting controlling genes for complex traits and thus serve as main coping strategies. To differentiate complex traits is of great importance for biodiversity research and studying genes, and is a key approach to understand the underlying mechanism of gene controlling. The existing methods, however, are not mature and perfect, which therefore have brought much difficulty in effectively differentiating complex traits in the field of biogenetics. Since complex traits can be effectively described as growth curves, methods based on growth curves in recent years are common way to differentiate complex traits, among which functional mapping (FM) is a representative method. although FM method has been one of the best approaches for differentiating complex traits over the past decade, it has been so far confined to deal with those where growth curve is monotonic. Earliness index (E-index) method emerges as required and successfully solves the non-monotonicity situation. Moreover, it is able to deal with any type of biological complex developmental process. Based on the principle of E-index, E-index application (EIA) analysis tool was developed, which provides a good platform for potential genetic researchers and scientists, helping them in several aspects, including data acquisition, drawing growth curves by dynamic data visualization technology, data processing and result retrieval. Results from simulation experiments show that EIA analysis tool is efficient, real-time and accurate, being a powerful tool to differentiate complex traits.
Detection of genetically modified organisms (GMOs) is an important part of the safety supervision of GMOs. There is a need for a rapid method of detecting genetically modified components suitable for large-scale screening, in order to expand the scope of genetically modified organism's safety monitoring. Immunochromatography test strip (ICTS) is a kind of fast immunoassay technology and it is one of the commonly used fast detection methods. Due to its simple operation, small quantity of sample, rapid detection, and low cost, etc., ICTS has been widely used in many fields, such as medicine, food and environment. The composition and structure, detection principle, and immune markers of ICTS were introduced, and its research progress in the detection of GMOs was summarized.
Next-generation sequencing technology plays an important role in elucidating the complex and highly repetitive genome structure, and the relationship between DNA sequence and genomic structural variation with the important agronomic traits. The NGS technology in crop genome research, including the development of the crop genome sequencing NGS technology, NGS and the transcriptome analysis, genome-wide association map and NGS, SNPs development and forecasting breeding, etc.were reviewed. It aims to provide a theoretical basis for crops genomic research.
Phytoferritin is an important iron regulatory protein in plants. Many studies have shown that phytoferritin has strong connection with oxidative stress resistance. Phytoferritin can not only defense the oxidation toxic from high iron, it also play a role in a lot of resistance to oxidative and environmental stress. The responses of phytoferritin to oxidative and environment stress are reviewed, to provide basis for the application of phytoferritin in biotechnology.
Cell drugs have their own characteristics and advantages which play the role that other drugs are difficult to play and have a good curative effect for cancer, autoimmune diseases and other illnesses. But in the process of research and development, there are ethical, safety, and other issues. To solve these problems, some solving strategies have been put forward, and the broad prospects of cell drug development are discussed. Besides,the latest cell drug clinical research and development situation are also introduced.
