Objective:This study was designed to express human MMP-2 C-terminal hemopexin-like domain (PEX) in E. coli, and then to test its biological effects on angiogenesis, as well as on the growth and metastasis of human breast cancer BICR-H1 cells. Method:PEX was cloned into prokaryotic expression vector pET28a (+) and was induced to express in BL21(DE3)-pLys by IPTG. PEX-His fusion protein, which existed in the inclusion bodies, was denatured by urea method, then was renatured and purified though Ni-NTA agarose beads. The purified PEX-His was further characterized for its biological effects on angiogenesis inhibition, as well as on the suppression of the growth and metastases of human breast cancer BICR-H1 cells by employing the CAM assay. Results: Human PEX expressed in E. coli was able to inhibit the growth of human umbilical vein endothelial cells in a time and dose dependent manner, and could effectively inhibit the CAM angiogenesis. Furthermore, the growth and metastases of BICR-H1 cells were suppressed significantly by injection of 10μg PEX through the vein of chicken embryos, and they were completely disappeared when 30μg PEX was administrated. Conclusion: Human PEX expressed in E. coli is an effective angiongenesis inhibitor. It can inhibit the growth and metastases of BICR-H1 cells, and it might be useful in the treatment of angiongenesis associated diseases such as cancer.
The Kringle 5 domain of plasminogen, which was previously shown to inhibit angiogenesis in vitro and vivo, is one of the most potent angiogenesis inhibitors described to date.The Kringle 5 gene was amplified from pET32a-K5 by PCR, and then was inserted into pET15b to construct a prokaryotic vector pET15b-k5.The target protein was highly expressed in E.coli BL21(DE3) and was mainly present in the precipitation observed by SDS-PAGE. The protein was purified by ion-exchange chromatography (SP Sepharose Fast-Flow) using the NaCl linear gradient strategy. The analysis results of SDS-PAGE showed that the purity of obtained Kringle 5 was about 98%. After desalting with size-exclusion chromatography, it was found that it inhibited the blood vessel growth of chick embryo chorioallantoic membrane effectively. This simple, effective method may be applied in the future in mass production of Kringle 5 and lay a solid base for detailed evaluation of bioactivities of Kringle 5.
To further modify the recombinant human kallikrein 1(hK1) for higher activity, new form of recombinant human kallikrein 1(hK1-L-Fc) was constructed by fusion of the human kallikrein1 gene and the coding sequences for Fc fragment of human IgG1 with a flexible linker peptide. The fusion gene hK1-L-Fc was constructed by PCR, then inserted into expression vector pcDNA3.1, and expressed in Chinese Hamster Ovary cells. The chimeric protein was purified by Protein A affinity chromatography, and further analyzed by SDS-PAGE、Western blotting、MALDI-TOF-MS and HPLC. The bioactivity of fusion protein in vitro was determined by the reaction with its substrate. The results showed that recombinant expression vector pcDNA3.1-hK1-L-Fc was constructed successfully and the cell lines stably secreting fusion protein were obtained; The expression output of the fusion protein was higher than 0.7 mg/L in batch culture; The purity of the protein with about 60 kDa molecular weight was higher than 95%, and the biological activity of the fusion protein was more than 9.2 U/mg, which increased more than 18% than that of hK1-Fc fusion protein.
Objective: To investigate the inhibitory effects of recombinant interference plasmid pshRNA-COX-2 on the growth and angiogenesis of human liver cancer cell Hep3B subcutaneous xenograft tumors in nude mice. Methods: The COX-2 mRNA and protein expressions were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, the VEGF mRNA expression was detected by RT-PCR after Hep3B cells were transfected with plasmid pshRNA-COX-2 and selected. Selected Hep3B cells were transplanted into the subcutaneous tissue of nude mice. Xenograft tumor volume was measured every three days and growth curves of tumors were drawn. The mice were killed after four weeks. The expression of COX-2 protein and microvessel density (MVD) in xenograft tumors were observed by immunohistochemistry. Results: Compared to the Hep3B cells without transfected by plasmid pshRNA-COX-2, the inhibition rates of COX-2 mRNA and protein expressions were 65.3% and 52.8% respectively in the pshRNA-COX-2 group (P<0.05), the inhibition rate of VEGF mRNA expression was 56.5% in the pshRNA-COX-2 group (P<0.05). The tumor volume in the pshRNA-COX-2 group was apparently smaller than that of the pshRNA-HK group and the untreated group (P<0.01). The COX-2 score and MVD in the pshRNA-COX-2 group were significantly lower than that of the pshRNA-HK group and the untreated group (P<0.01). Conclusion: The plasmid pshRNA-COX-2 can significantly inhibit the growth and angiogenesis of human liver cancer cell Hep3B subcutaneous xenografts in nude mice through inhibiting COX-2 expression.
Objective:To explore the effect of dendritic cell modified by PEG10 gene for liver cancer. Methods:Dendritic cells originated from the HLA-A2+ PBMCs were transfected with recombinant adenovirus containing PEG10 full length cDNA, and the modified DC were utilized to generate specific cytotoxicity T lymphocytes (CTL). The activity was analyzed by standard 51Cr releasing assay and ELISPOT. Results:The transfected DCs could elicit specific CTLs, which could secrete IFN-γ and lyse liver tumor cells. Conclusions:The PEG10 recombinant adenovirus transfected DCs may provide a new therapeutic strategy for hepatocellular carcinoma.
The TAT protein transduction peptide was enriched in basic amino acids and encoded by the HIV-1 virus. Previous studies have revealed that it could safely and efficiently mediate various heterologous biological macromolecules across a variety of biomembranes, such as the plasmid membrane and the blood-brain barrier et al. To further study its function in mediating heterologous proteins transduction in nematode in vivo, the prokaryotic expression vector pET28b-EGFP and pET28b-TAT-EGFP were constructed and induced by IPTG (final concentration 1 mmol/L), followed with the analysis on the expressed protein by fluorescence microscopy, SDS-PAGE and Western blot. Subsequently, the bacterial cells were coated to the LB medium and directly fed to the nematodes followed with capturing the image at 48 h. Results showed when fed to the nematodes for 48 h, the TAT-EGFP fluorescence signals were clearly distributed in the intestinal cells of the worm, while the EGFP fluorescence signals were mainly distributed in the intestinal cavity of the animal. Furthermore, the cellular morphology of TAT-EGFP had no distinct change compared with the EGFP group and controls. Taken together, the data suggested the TAT protein transduction peptide could mediate heterologous protein expression in C.elegans and provided an alternative approach for development of new drug transporter.
Protein SRPK3 acted as a crucial element of transcription pre-initiation complex, which play an important role in regulation procession of gene expression. In order to explore the genetic characteristic of SRPK3 in pigs. SRPK3 gene came from Yorkshire a pig was cloned by RT-PCR yet coding sequence was completed. The distribution determination of mRNA came from ten Yorkshire pigs and Duroc pigs in heart, muscle, liver, kidney, lung stomach, small and large intestine, spleen, brain was finished by Real-time PCR by age one day and 30 days. Expression test of gene SRPK3 was implemented in a skeletal damage model during the period of skeletal muscles development. Sequence analysis of an mRNA fragment with a length of 1 708bp in gene SRPK3 of a Yorkshire pig revealed a full coding region, 1 701bp, which coded 656 AAs including two S_TKc domains. The sequence of the porcine protein SRPK3 shared high similarity with its homolog from human and bovine, and they were closed in the Phylogenetic tree. PCR-SSCP test shows: On the Sixth exon (CDS: 629, CDS: 653) get A→G,T→C, which change the amino as Pro → His,Ile→Thr, on the ninth exon (CDS: 1059) get a G → A, but no change of amino. Both breeds specific expression and tissues specific expression were detected by RT-PCR, however high expression was mainly detected in skeletal muscle and heart. The quantity of mRNA of gene SPRK3 in the period of skeletal muscle destruction and repair, which showed gradually to be increased in different stage. Because of it’s expressed in skeletal muscle and heart mainly, and gradually be increased in different stage of injury and repair process.SRPK3 may be related to skeletal muscle cell development.
Objective:To study the effects of NH4+ on biosynthesis of L-tryptophan. Methods:Fed-batch fermentation of E. coli TRTH with addition of ammonium sulfate carried out in 30-Liter fermentor and biomass, yield of L-tryptophan, consumption rate of glucose, plasmid stability were measured. The concentration of acetate, NH4+, lactate and pyruvate were investigated by BioProfile 300A Nova and high performance liquid chromatography (HPLC). The metabolic flux balance model of L-tryptophan synthesis by E. coli was established. Based on this model, the practical metabolic flux distribution of E. coli TRTH were determined with the linear program planted in MATLAB software. Results:During the fed-batch culture, E. coli TRTH was able to maintain higher growth rate at exponential phase, finally, the biomass and the yield of L-tryptophan were increased by 51.07% and 46.54% respectively by controlling the concentration of NH4+ less than 120 mmol/L. As the increase of the concentration of NH4+, the concentration of pyruvate, lactate and acetate were slightly increased, but cell plasmid stability was decreased. Data indicated that EMP pathway and TCA cycle were decreased by 7.31% and 22.04% respectively, PP pathway was increased by 7.14% compared with addition 5g/L of ammonium sulfate at 14h during the fed-batch culture. Conclusion:High concentrations of NH4+ terminated the cell growth, decreased the consumption rate of glucose and inhibited the L-tryptophan production. Controlled the concentration of NH4+ less than 120 mmol/L, the biomass and the yield of L-tryptophan were significantly increased, which accessed high cell density cultivation.
In the study of photobioreactor, gas distributor has great influence to the growth of microalgae, especially in the bubbling column reactor.The effect of the gas-flow rate and CO2 concentration on the biomass, chlorophyll a, and lipid accumulation of Chlorella vulgaris LICME002 in the 5L bubbling photobioreactor with a alumina gas distributor. The results showed that the 3% CO2 is the optimum condition for biomass, chlorophyll a, oil accumulation. When the CO2 concentration exceeded 6%, the algae's parameters decreased significantly. With the analysis of the algae's parameters at 0.1vvm,0.4vvm,0.7vvm, 1.0vvm, and the gas-flow rate 0.4vvm is the best one. Results showed that the optimum gas-flow rate and CO2 concentration, the microalgae biomass can achieve 1.52 g/L, oil content achieved 31.5%.
A fermentation system composed of a stirred tank followed by four-stage packed tubular bioreactors in series was set up to evaluate very high gravity ethanol continuous fermentation using Saccharomyces cerevisiae. Polyurethane and wood chips were used for cells immobilization in tubular reactors, and ethanol productivity in later reactors were maintained at high level by high bilomass content even the viability and ethanol productivity of cells were inhibited by high ethanol content. The system ran at 0.02h-1 and fermented very high gravity medium containing 280 g/L glucose to produced 15.4 % v/v ethanol. It was also proved that dilution rat should reduce by new reactors added to higher final ethanol content under the lower dilution rat level.
The ethanol fermentation ability of SA-PVA-SiO2 immobilized Saccharomyces cerevisiae and free S. cerevisiae was compared. In addition, batch fermentation was used to test the stability of ethanol fermentation of immobilized S. cerevisiae. The results showed that, the fermentation speed of SA-PVA-SiO2 immobilized S. cerevisiae was faster than free S. cerevisiae; the fermentation stability of the immobilized S. cerevisiae was good, the volume fraction of ethanol remained 3%~3.5 ％ when cultured at 30 ℃ for 24 hours using rubber plug and the shape of balls were still intact, not sticky afte 14 batches continuous fermentation; The internal immobilized environment was very conducive to the anaerobic fermentation to produce ethanol by SA-PVA-SiO2 immobilized S. cerevisiae.
A novel, simple and highly efficient process for purifying vanadium bromoperoxidase from Corallina officinalis is developed, which is consist of two steps: crude enzyme extraction was heated at 70℃ for 2h, and then was ultrafiltered with PVDF membrane. The ultrafiltration conditions were optimized, and the optimized conditions are: MWCO=100kDa, 0.02MPa pressure, 600r/min stirring, 0.12mg/ml initial protein concentration and pH=6.0. The crude extraction purified with heat treatment and then ultrafitration results a 21-fold purification, with a 96% overall yield and 212U/mg specific activity.
The stem cell has become an ideal supply of cells for tissue engineering and cellular therapies for its’ capacity of long-term self-renewal and multipotency-the ability to differentiate into one or more specialized cell types properties. These approaches require a readily available source of stem cell outside a living body. But the stem cell's microenvironment hasn't been clearly defined for its' multiplicity and complexity, and the conventional culture cell method's limitation also dedicate for it. The stem cell's self-renewal or differentiation fate couldn't be controlled outside a living body with current stem cell research method. As the stem cell is very sensitive for the microenvironment change, precisely mimic and control the stem cell microenvironment that control the stem cell's self-renewal or differentiation fate has become the difficulty in stem cell research. With the introduction of microfluidics into cell culture technology, it is possible that mimic the stem cells’ in vivo microenvironment in vitro. For it can culture stem cell in 3D microenvironment; precisely control the various factors and study their influence on the stem cell fate. Furthermore microfluidic chip can also be made transparent and monitor the stem cells real time by imaging.In the microfluidics and the blood vessel endothelial progenitor cell, review the microenvironment that the stem cell needed and enumerate the advantages of microfluidic technology, summary some successful research findings for the stem cell research inside microfluidics. And the prospect of this technology for the stem cell microenvironment and for other applications was made.
Recombinant DNA vaccines, subunit vaccines and synthetic peptide vaccines have sprung up one after another in recent years. These vaccines are characteristic with high purity, specificity, but they can not induce efficient immune response because of small molecule and low immunogenicity. Therefor, there is demand for safe and non-toxic adjuvants able to enhance the protective immune response. Recent advances in basic immunology have elucidated how early innate immune signals can shape subsequent adaptive responses, and this coupled with improvements in biochemical techniques, has led to the design and development of more specific and focused adjuvants.The type and mechanism of adjuvants, the progress of study on innate immunity and also explores future directions of adjuvant development are reviewed. All these could provide some references for the study on highly active, low toxicity adjuvants.
Rapid nucleic acid amplification in vitro is a revolutionary technology developed since 1983. It has been applied widely in modern agriculture, medical and food science etc.. Such technology has provided a great tool for gene manipulation and functional genomics. In vitro nucleic acid amplification is usually carried out by a thermal stable enzyme, such as Taq polymerase, and requires thermal cycle equipment. The invention of isothermal amplification has improved the traditional PCR method and in some way simplified the reaction procedure. Recombinase-aid amplification (RAA) was introduced. RAA is the latest isothermal amplification technology which may be carried out under the room temperatures. The distinctive merit of RAA is mimicking in vivo T4-phage DNA amplification complex and the whole reaction is super fast and specific. Moreover, it can also be used for digital real time quantitative analysis.
Human respiratory syncytial virus (RSV) is the leading viral agent of serious respiratory disease in infants and young children worldwide, infecting nearly all children one or more times by age 2. An intranasal live-attenuated RSV vaccine mimics natural infection and presumably induces the same broad array of local and systemic immunity, including innate and cell-mediated immunity as well as mucosal and serum antibodies. The intranasal route is albe to escapes the immunosuppressive effects of maternal serum antibodies. Additionally, the immune-mediated enhanced disease, induced by nonreplicating killed RSV vaccine, is not associated with attennated RSV vaccine candidates. A number of live-attenuated RSV vaccine candidates have been developed by conventional methods. However, there are many advantages for developing attenuated strains by reverse genetics.The application of reverse genetics to the live-attenuated RSV vaccine is reviewed.
Ovine adenovirus 287 has a unique genome organization with a length of 29 576bp. The genome is characterized distinctively by a high A/T content (66.4%), the lack of a typical El region, and a long redundant sequence. Three nonessential regions of the viral genome, where 6.3kb foreign genes can be inserted theoretically, have been identified. OAdV287 also has unique fiber and penton, where lacks the integrin-binding domain in penton or any other capsid protein. OAdV287 can infect a wide range of human and animal cell lines, but failing to replicate in non-ovine cell lines. In addition, OAdV287 vector can circumvent the pre-existing human adenoviruses immunity, and so have an efficient transfection. Currently, OAdV287 has become one of the research focuses among the animal adenoviruses vectors.
Lactoferrin is a key component of the mammalian immune defense system. It shows a multifunction involving in iron regulation, broad spectrum of antimicrobial activity, anti-inflammatory activity, anti-tumor activity and immunologic enhancement. But its potential nutritional and therapeutic applications have been limited due to the high cost for extraction and purification of functional lactoferrin from milk. Recombinant expression would be a feasible approach to produce active lactoferrin in large scale. The advances of lactoferrin recombinant expression in E. coli, yeast, insect, plant and mammalian cells were reviewed.
Insoluble, inactive inclusion bodies are often formed during prokaryotic expression of foreign genes. The formation of these deposits represents a major obstacle for the production of biologically active proteins for further investigation and application. However, after solubilization, isolation and refolding under suitable condition, inclusion bodies can convert to active proteins in vitro. So far, about eighteen snake venom proteins of eleven species in Viperidae and Elapidae(including metalloproteinases, phospholipase A2s, β-Bungarotoxin, cardiotoxins, serine proteases, nerve growth factors, C-type lectins) have been expressed in E. coli and successfully refolded by means of dilution, dialysis or chromatography. The refolding techniques for inclusion bodies of prokaryotically expressed snake venom proteins is summarized.
Heat shock protein 30 is one of small Heat Shock Proteins (sHSPs), and is being widely investigated in fungi. Genes encoding heat shock protein from different fungi have been cloned and tested. Studies on HSP30 was mainly focus on the expression of stress leves and transcriptional regulation,and the synthesis mechanism of HSP30 under stress response is still unclear. The research progress of HSP30 and its application prospect is reviewed.
