Objectives: We aimed to construct an eukaryotic expressive vector pCMV-Luciferase-IRES2-EGFP which contains luciferase and enhanced green fluorescent protein(EGFP) and steadily screen Hepa1-6 cell line transfected with this recombinant vector,with the purpose of stably labeling and in vivo tracking the hepatoma cells in orthotopic hepatoma model established using this cell line. Methods: pGL3-Basic vector was digested by Xba I,end-repaired by Klenow Fragment and then redigested by Xho I to acquire the luciferase gene,which was ligated with the larger fragment of pIRES2-EGFP vector through Xho I/Sma I double digestion to construct the recombinant vector. Authenticated by endonuclease digestion and sequencing,the recombinant product presented the correct sequence. The expression of EGFP and activity of luciferase of Hepa1-6 cells transfected with this vector were,respectively,dectected by fluorescent microscope and dual-luciferase reporter assay and LuminaⅡ imaging system.Confirming the expression of the vector in the cell line, we inoculated this cell line orthotopically in the liver of C57BL/6 mouse to establish a hepatoma model.The growth of the hepatoma was consecutively monitored in vivo using LuminaⅡ imaging system and both paraffin sections(HE staining) and frozen sections (immunofluorescent staining) were prepared from the ex vivo tumor tissue to observe the pathological features and EGFP expression of the hepatoma cells. Results: Hepa1-6 cell line expressing the recombinant vector pCMV-Luciferase-IRES2-EGFP(EGFP-Luc-Hepa1-6) was successfully constructed.Using this cell line,an orthotopic hepatoma model in mice was established with the capability of in vivo tracking and ex vivo labeling the hepatoma cells. Conclusions: The EGFP-Luc-Hepa1-6 cell line was constructed successfully and the orthotopic hepatoma model established using this cell line can satisfy the needs of detecting the progression of the hepatoma both in vivo and ex vivo.
Objective: To investigate the biological effect of S100A9 on human hepatocellular carcinoma cell line HepG2 and the relevent mechanism. Methods: Immunohistochemiy and Western blot assay were used to detect S100A9 expression in human hepatocellular carcinoma (HCC) intratumoral and peritumoral tissues. Prokaryotic expression system was used to prepare recombinant protein GST-S100A9. HCC cells HepG2 and live normal cells L02 were treated with GST-S100A9, then MTT assay was used to study the cell proliferation, and invasion assay was used to study cell invasiveness. Western blot assay was used to detect advanced glycation end products (RAGE) expression in HepG2 cells and L02 cells. Results: The expression of S100A9 was higher in HCC intratumoral tissues than that in peritumoral tissues. GST-S100A9 promoted the proliferation and invasion of HCC cells HepG2, but no effect on live normal cells L02. The expression of the receptor for RAGE in HepG2 cells was higher than that in L02 cells. Treatment with RAGE blocking antibody abrogated the S100A9-promoted proliferation and invasion of HepG2 cells, demonstrating that these biological effects were involved in RAGE. Conclusion: S100A9 promotes the proliferation and invasion of HepG2 cells involving RAGE.
The site-direct modification conditions for mutant CNTF-C17 with a molecular weight of 40kDa amaleimide-PEG (mPEG-MAL) was optimized. The mono-PEG-CNTF-C17 was obtained through ion-exchange chromatography and characterized by CD and FL. Experimental data showed that the optimal ratio of protein to PEG was 1:3 and reaction condition was at 4℃ for 12h in Tris-HCl buffer. At the optimized condition, the PEGylatoin yield of mono-PEG-CNTF-C17 can easily reach more than 90%. By anion exchange chromatography mPEG40K-MAL-CNTF-C17 could efficiently separated from the modified mixture with a purity of 98% and a recovery of 78.1%. FL and CD spectra illustrated that mono-PEG-CNTF-C17 has almost the same second and tertiary structure as the natural protein. After PEGylation, the specific activity of CNTF-C17 reduced to 6.51 × 105IU/mg measured by TF-1.CN5a.1 cell. But the half-life in vivo was prolonged 30 times. This study provided a foundation for developing new long-acting CNTF product.
Nonalcoholic fatty liver disease (NAFLD), one of the most prevalent chronic diseases worldwide, is associated with accumulation of fat in liver and is a manifestation of metabolic syndromes (including obesity, diabetes, and insulin resistance). HepG2 cells are selected to establish a model in vitro about hepatic steatosis and to identify the transcription factors (TFs) that change their binding affinity to DNA in response to hepatic steatosis. HepG2 cells are incubated with 2mmol/L oleic acid for 24 hours. Oil-Red-O staining is done to show triglycerides (TG) and assess the extent of hepatic steatosis. The nuclear extraction from each group is separated from the cytoplasmic extraction and the transcription factors in nuclear extraction are enriched by a concatenated tandem array of consensus transcription factor response element (cat TFRE). By combination of catTFRE and an in-depth analysis of proteomics expression profiling, the dynamic description and quantitative analysis of the transcription factors' DNA binding activity changes are shown. Then the functions of the altered transcription factors are analyzed by IPA(integrated pathway analysis). 170 TFs are identified in the control groups and 190 TFs are identified in the oleic acid treated groups. 208TFs are identified in all experiments. In the 208 TFs, DNA binding activity of 67 TFs are up-regulated obviously and 34 TFs are down-regulated obviously. The DNA binding activity of MLX and MLXIPL, which play important roles in glycometabolism, down-regulated obviously. The DNA binding activity of NF-κB1 is up-regulated obviously and suggested that the NF-κB-mediated inflammation is activated.The results of IPA revealed that NRF2-mediated oxidative stress response is activated in response to the oxidative stress and the progresses of gene expression, cell proliferation, cell differentiation and cell cycle are increased. The conclusion is that after treated with oleic acid, although the balance between glycometabolism and lipid metabolism is broken, inflammation is activated and oxidative damage is caused, HepG2 cells tend to take actions like increasing gene expression, cell proliferation and activating oxidative stress response at the transcription level and finaly keep cells survival.
As a response regulator in the two-component signal transduction system (TCST) of Staphylococcus aureus, AgrA, which transmits signals to downstream promoter and then regulates genes expression and transcription, plays an important role in the pathogenesis of S. aureus. A expression vector using Restriction-free (RF) cloning was constructed. In order to monitor the protein expression level in real time, a C-terminal green fluorescent protein (GFP) was fused to AgrA to serve as a reporter. Firstly, BL21-(DE3)-PlysS was screened as the best host cell by single-factor experiment. Secondly, combined with the Box-Behnken designed response surface method test, the cultured conditions which include culture time、RPM and IPTG concentration were optimized. The optimum conditions selected are as follows: induction time is 22h, speed is 222r/min, IPTG is 0.5mmol/L. Then, the expressed AgrA were purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) with the yield of AgrA is 5.56mg/L. Finally, the activity of AgrA was assessed by using non-radioactive electrophoretic mobility shift assay (EMSA) based on AgrA protein LytTR region. In short, A strategy through which response regulator protein AgrA could be expressed in E. coli efficiently and solublely. It not only establishes the foundation for the two-component signal transduction system in vitro studies, but also provides a viable reference for other response regulator proteins which need soluble expression, separation and purification.
To improve the absorption and tolerance of HCHO in plants, the plant expression vector pk2-PrbcS-*T-CAT was constructed using Arabidopsis thaliana CAT (catalase) coding region. Overexpression of CAT in wild-type (WT) tobacco chloroplasts produced two transgenic lines (C1, C3) which had different expression quantity. Using 10ppm, 20ppm and 40ppm gas HCHO treated WT and transgenic tobacco, and then analyzed the HCHO absorption efficiency and tolerance of CAT transgenic tobacco. The results showed that the absorption quantity of gas HCHO by transgenic tobacco was higher than wild type (WT), and with the increasing concentration, total soluble glucide, total protein and total chlorophyll content in leaves of the transgenic lines were significantly higher than WT. The oxidative damage index of H2O2, malondialdehyde (MDA) and protein carbonyl (PC) which content in transgenic lines leaves were significantly lower than the WT. The PM H+-ATPase and hydrogen pump activity of transgenic tobacco (C1) was 3 times and 2.5 times than WT after 40 ppm HCHO treatment. Stomatal conductance of C1 is 2.67 times than WT. These results indicated that overexpression of Arabidopsis CAT could reduce oxidative damage in tobacco cells which induced by HCHO, and improved the ability of tobacco to HCHO detoxification and enhanced the absorption and resistance of tobacco to HCHO. It was revealed that the stomatal conductance affected by PM H+-ATPase and hydrogen pump activity when gas HCHO stressed tobacco, which might be one of the causes of higher HCHO absorption efficiency of transgenic tobacco.
Previous studies, a recombinant E.coli WL204 was constructed,which contained a chromosomal integrated ldhL gene and possesses the capability of homofermentation of L-lactic acid using xylose as substrate. Wastepaper was selected as feedstock to evaluate the fermentative production of lactic acid by recombinant E.coli WL204.The wastepaper chips were treated with 1:6-1:14(w/v)H2SO4, and then incubated 60 h at 50℃ with cellulase. The resultant hydrolyzates were detoxified with dried Ca(OH)2, which could remove most of furfural and HMF. Fermentation of the detoxified hydrolyzate of wastepaper with E.coli WL204 produced 31g L-lactic acid from 100g dry wastepaper and the lactic acid yield coefficient was 79%.These results demonstrated that WL204 has the potential for homofermentative production of L-lactic acid using cellulosic biomass derived substrates.
As a gene delivery system, Lentiviral vector (LVV) has been being increasingly used in gene therapy trials, and drawn large attention in recent years. Yet, transcriptional read-through (TRT) is one of potential risks in its application. For the purpose of improving the detection methods of TRT, and making the results intuitive and accurate, a codon optimized reporter gene, LacZ, was placed downstream to the 3'- LTR of the proviral vector to simulate its state in chromosome. Upon the transfection followed with LacZ staining, the cells with TRT phenomenon would be stained blue, and the TRT rate would therefore be visualized at the translational level. Correlating well with the outcomes of qRT-PCR, the established methodology could reflect the feature of TRT, which paves a way for improving LVV biosafety for clinical applications.
To solve the problem that additional primers could be inserted at the mutation site when use QuickChange site-directed mutagenesis methods to mutate some specific sites, QuickChange method was modifed as followed:A pair of complementary primers containing mutations site were synthesized, and single primer PCR amplification was done respectively. The two PCR products from single amplification were mixed and denatured and renatured followed by addition of restriction endonuclease Dpn I. The product was transformed into competent E.coli DH5α, and positive clones were screened for DNA sequencing. Several sites in amorpha-4,11-diene synthase(ADS) gene that could not be successful mutated using tranditional method had been successfully mutated with this method without any additional primers. So this novel method was practicable and could solve the problem of extra primers insertion in QuickChange site-directed mutagenesis. This also indirectly proved that additional primers insertion was caused by putting two primers in one reaction system.
Glucagon-like peptide-1 (GLP-1) is a potential therapeutic drug for type II diabetes, mainly because of the stimulatory effect on insulin secretion under condition of high blood glucose. We used PCR to obtain a recombination gene AGGH, two GLP-1 (GLP-1A2G) mutants were connected in series and then fused to the N terminal of human serum albumin,and alanine was added at the N terminal of GGH. The fusion gene was inserted into pGAPZaA plasmid and expressed by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The engineered strain was constructed and the recombinant P. pastoris successfully expressed the fusion protein AGGH. The yield of AGGH reached 68 mg/L after 72 h fermentation in a flask, using glucose as the optimal carbon source. Fed-batch fermentation was investigated in a 5 L bioreactor, and the expression level of GGH reached 238 mg/L in 52 h. The fusion protein AGGH was purified in four steps, and the final purity was 95.8%. The bioactivity of AGGH was the same as that expressed in P. pastoris by the AOX1 promoter. This study described an efficient way to express AGGH fusion protein in P.pastoris using GAP promoter, fermentation was easy way to control without carbon source change than AOX1 promoter-controlled GGH expression and fermentation time was 20 h less than AOX1 promoter-controlled GGH expression.
New human serum albumin (HSA) specific binding 7-amino-acid peptides were screened and identificated by using phage display technology. The affinity of the fusion polypeptide, comprising the screened 7-amino-acid peptides and pituitary adenylate cyclase activating peptide 27 (ML-PACAP27), were quantitatively determined,so that it was determine that the screened 7 peptides (ML1 or ML2) in fusion proteins including other peptide or protein drugs still remain the high binding activity for HSA. Using M13 phage-displayed 7-mer peptide library, the affinity effect of ML1 or ML2 and HSA were preliminarily identified through enzyme-linked immunosorbent assay (ELISA) after four screening rounds and DNA sequencing of the screened clones. Affinity constants of ML1-PACAP27 or ML2-PACAP27 and HSA were assayed by surface plasmon resonance (SPR). The experimental results showed two new 7-amino-acid peptides ML1 and ML2 were obtained, and their amino acid sequence is LKSCKPL (ML1) and SLKSHAL (ML2). The ELISA results showed the screened clones containing ML1 and ML2 can bind to HSA, and the affinity effect of ML2 for HSA is higher than that of ML1. The SPR results showed the dissociation constants of ML1 and ML2 for HSA is respectively 8.1×10-6 and 3.7×10-6 mol/L,and the affinity of ML2 and HSA is higher than that of ML1. In the study, two new HSA-specifically binding 7-amino-acid peptides that were screened and identificated may be used for the fusion expression or design of HSA-conjugated molecule drugs with long-acting effect, which can provide the new tools for extending the half-life of molecule drugs and the development of the novel drugs.
The genes encoding tet repressor proteins and Ketogulonigenium vulgare sorbose dehydrogenase operator which inserted operon teto were synthesized by overlapping PCR. The shuttle vector of tetracycline induced expression with broad host plasmid pBBR1MCS-5 was constructed and transformed Ketogulonigenium vulgare. The resistance of kanamycin was regulatively expressed after getting the positive recombinant strains. The results show that the cultivation of recombinant strains could grow in the medium containing kanamycin where added 0.4mg/ml tetracycline inducers after 2 hours, while they can't grow in the medium containing kanamycin where didn't add tetracycline inducers. The optimal induction of tetracycline concentration determined was 0.6mg/ml.
Purine nucleosides and their derivatives play an very important role in food and pharmaceutical industry. In recent years, they have gained further importance because of their beneficial effects, related to their antioxidant, neuroprotective and immunomodulatory properties. However, It is time consuming and low efficiency to get high-performing strains through mutation screening techniques. Moreover, some of these strains have disadvantage of genetic instability. With the better understanding of regulation mechanism of microorganism, metabolic engineering has been widely applied to improve the production of purine nucleoside products. In this article, the purine biosynthetic pathway and regulation mechanism are discussed, and progress in metabolic engineering of purine nucleosides and their derivatives is also reviewed. Meanwhile, the current problems and future research direction are proposed.
The differential expression of genes is closely related to biological characters and function of tissue and cell. With the extensive study on the differentiation of tissue and organs and the growth and development of cells and individuals, the validation of differentially expressed genes are getting more and more important. Thus, a series of validating techniques of differentially expressed analysis were established on the basis of nucleic acid hybridization and PCR, such as Northern blot and fluorescence quantitative PCR. The differentially expressed genes were validated of different treatment, different organs and different development period using these methods, which laid a solid foundation for the gene functional analysis. With the fast development of the detecting methods, the validation methods of differential expression of genes has developed from qualitative to quantitative, from tedious and complicated to simple and rapid, from requiring a large number of initial RNA to only a small amount of RNA, even the fluoresce quantitative PCR method for single cell has been established, and the method is to be more efficient, accurate and low cost. However, The validation for the samples difficult sampling like germ cell and less amount of RNA containing like sperm is still a great challenge for the current method. The validation methods of differential expression of genes from the perspective of the development process was reviewed. Reference to the detedtion of samples dificult sampling and less amount RNA containing was provided.
FMDV is a small, nonenveloped, positive-strand RNA virus belonging to the genus Aphthovirus within the family Picornaviridae. It contains about 8 400 nucleotides. Many studies have found that it is very important for infection that FMDV recognizes its receptor on cellular surface and enters host cell. Endocytic pathways of which FMDV enters host cell by depend on receptors of which FMDV uses to infect host cell. In recent years, there are a lot of work about the invasion of FMDV to host, explaining many problems about infection mechanism of foot and mouth disease, which provides a strong important base for the problem on actual production. The purpose of this paper is to summarized the previous studies, in order to providing reference for further study of pathogenic mechanism about foot-and-mouth disease virus and to exploring more effective prevention and control measures.
Hydrogenase enzymes, which catalyze the formation and dissociation of hydrogen are heteromeric metalloenzymes. Mature hydrogenases are usually highly sensitive to oxygen,and the pro-enzymes are not active unless they are modified by a complicated post-transltational maturation process which involves synergized work on catalytic center of the enzymes by related chaprons. Catalytic mechanisms of hydrogenases also plays pivotal role in development of valuable oxygen-resistant biocatalysts for bio-hydrogen production and synthetic hydrogenase mimics applied in green battery industry. Recombinant enzymes are therefore indispensable for enzymes' structural studies, since acquisition of native enzymes is extremely difficult, and, in some case impossible. This review aims to summarize and analyze recent progress in studies using native or foreign hosts to achive successful expression of recombinant enzymes that are either iron only or NiFe containing. Furthermore, the enzymatic features were systematically compared between native and recombinant proteins, and the likely solutions for future works in this area were also proposed.
