Objective: To establish transgenic mouse model of PTEN (phosphatase and tensin homolog does on chromosome ten), one of the most studied tumor suppressor genes in cancer field, and preliminarily analyze the phenotype of PTEN transgenic mouse model. Methods: The targeting vector of PTEN transgenic mouse model was designed through bacterial artificial chromosome (BAC) carrier system, which can protect the inserted genes from position effect and ensure high expression of the inserted gene. PCR was used to identify the genotype of F0 mice and offspring. Positive transgenic mice in F0 generation were crossed with wild type mice respectively to screen the stable transgenic mice line. At the same time. PTEN protein expression level was detected both in the embryonic and adult mice respectively, embryonic fibroblasts (mouse embryo fibroblast, MEF) from PTEN transgenic mice and the wild type littermates were used to be detected and evaluated PTEN protein levels by Western blotting, and clone-formation assay was used to determine the MEF proliferation ability. Adult mice tissues of the high expression line were used to be a verification. Once confirmed PTEN was really over expression in the transgenic mice, weight of mice from the high expression transgenic line were recorded and analyzed from 3 to 12 weeks. In addition, the cell size of liver, spleen and lung and abdominal fat were compared between PTEN transgenic and wile type MEFs. Results: PTEN transgenic mouse model was successfully constructed by taking advantage of BAC carrier system, genotyping the offspring of PTEN transgenic mice showed it can breed normally. Western blotting showed that PTEN expression levels were significantly higher in transgenic mice than in wild type ones, PTEN protein levels in embryonic fibroblasts cells (MEF) from transgenic mice were as 3 folds as that from the wild type littermates, PTEN protein levels were also significantly higher in transgenic mice tissues than that in wild type ones. Moreover, the body size of PTEN transgenic mice is obviously smaller in the body size than that of wild type ones and intra-abdominal adipose content is significantly reduced. However, after comparing the cell size in liver, spleen and lung between transgenic mice and wile type mice, results showed that cells exhibit a same size. Conclusion: By using BAC strategy,PTEN transgenic mouse model was successfully established. Through isolating MEFs and evaluating PTEN level, cell lines stably overexpressing PTEN were smoothly obtained, which are very important for the study of tumor suppressor gene PTEN in physiological condition.
Objective: To evaluate immunoreactivity and value of chimeric proteins that carry VP4 epitopes on the same insertion site using rotavirus VP6 as a vector. Methods: Two plasmids that carry genes of chimeric proteins carrying VP4 epitopes on the same insertion site using rotavirus VP6 as a vector were constructed by gene cloning and recombination; the chimeric proteins were expressed in E. coli cells, detected with anti-VP6 antibodies, as antigens inoculated in guinea pigs; and neutralization activities of antibodies in sera from chimeric proteins inoculated guinea pigs were determined. Results: the expressed chimeric proteins could react with antibodies against VP6 of rotavirus, could elicit production of antibodies in inoculated guinea pigs that reacted with vector protein VP6F, VP6 and VP4 of strain Wa virus, neutralized infectivities of Wa virus in MA104 cell. The results showed that chimeric proteins carrying different VP4 epitopes on the same insertion site retained its original backbone structure stability and had good immunoreactivity; results obtained are valuable for development of novel rotavirus vaccines.
Objective: To explore the effects and the relative mechanism of ginsenoside Rg1 on bone marrow hematopietic function in aging model rats, and provide theoretical and experimental evidences for searching effective natural medicine of antiaging or protecting hematopietic function. Methods: Male SD rats were randomly divided into normal group, Rg1 normal group, aging model group and Rg1 aging model group. Aging model group: the rats were administrated with D-galactose 120mg/kg,qd×42 by subcutaneous way; Rg1 aging group: the rats were also given D-galactose of the same dose and time as aging model group, and from the 15th day on, rats were treated with Rg1 20 mg/kg qd×28 by intraperitoneal way; Normal control group: The rats were injected with same volume saline qd×42 by subcutaneous way; Rg1 normal group: the rats were given saline with the same volume,qd×14, then received Rg1 20 mg/kg qd×28 by same way. After 2 days of finishing the treatment, peripheral blood was collected to detect the number of leukocytes,analyzes classification of leukocyte and advanced glycosylation end products (AGEs); The femur was taken to count the number of bone marrow mononuclear cells(BMMCs) in each femur, the proliferation of BMMCs was detected; Senescence-accociated β-galactosidase (SA-β-Gal) stain was analyzed aging BMMCs; multipotential hematopoietic progenitor (CFU-Mix) was cultured to observe the colony formation of BMMCs; the expression of senescence-associated protein P21 and P53 in BMMCs was assayed by Western blotting; peritoneal macrophage was extracted and detected the phagocytosis of the macrophage by colorimetric method. Results: Compared with the aging model group, Rg1 aging model group can significantly increase the number of leukocytes, regulate the percentage of granulocyte and lymphocytes, the proportion of CD8+T cells and CD4+T cells; the number of BMMCs in each femur; decrease the ratio of SA-β-Gal positive BMMCs,the amounts of AGEs;enhance the ability of BMMCs to form CFU–Mix; down-regulate the expression of senescence- associated protein P21 and P53;promote the phagocytic index of peritoneal macrophage. Conclusion: The hematopoietic cells are obviously induced senescence by treating with D-galactose, ginsenoside Rg1 has a significantly antiaging or protective effect on hematopoietic cells. It is suggested that the mechanism may be Rg1 protecting hematopietic cells by regulating p53/p21 signaling pathway.
Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild cloven-hoofed animals, and its causative agent is FMD virus (FMDV). The suckling mouse is an important experimental animal model for FMDV and integrin αvβ6 is an important receptor of FMDV. In order to further study the role of integrin αvβ6 in FMDV infection for suckling mice, two subunit genes of integrin αvβ6 from suckling mice were cloned and transduced the Chinese hamster ovary 677 (CHO-677) cell line to stably express both suckling mouse integrin subunits αv and β6 (CHO-677-mαvβ6). The presence of αvβ6 in CHO-677-mαvβ6 cell line was detected by PCR and indirect immunofluorescent assay (IFA) at the gene and protein levels, respectively. In order to analyze the susceptibility of FMDV to the cell line, the wild-type strain Asia1/HN/CHA/06 and O/BY/CHA/2010 were used to infect the cell line. Viral RNAs were detected by real-time quantitative RT-PCR. Growth curves of the representative strains in the cell line and its parental cell line were determined by TCID50 assay. The data showed that CHO-677-mαvβ6 cell line was more susceptible to FMDV than the CHO-677 cell line, indicating that CHO-677-mαvβ6 cell line was constructed successfully.
The aim is to realize the recombinant expression of human lysozyme-like protein 6 (hLyzl6) by the Pichia pastoris expression system and to investigate its enzymatic activity. The hLyzl6 gene was synthesized chemically according to the codon usage preference of Pichia pastoris and cloned into the expression vector pPIC9K. The recombinant plasmid was linearized and transformed into competent Pichia pastoris GS115. After screening by G418 plate, the yeast strain was cultured and induced with methanol. The recombinant protein was purified by chitin affinity chromatography and subjected to activity assay. The results showed that the lytic activity in the culture supernatant reached the peak after 72 h of induction and the existence of the recombinant protein was confirmed by SDS-PAGE. The recombinant hLyzl6 exhibited bactericidal activity against Micrococcus lysodeikticus by using turbidimetric assay and had the activity of 54 700 U/mg. The optimum temperature and pH for hLyzl6 were 40℃ and 5.5 respectively and Cu2+ was found to have strong inhibition effect on hLyzl6. The EC50 value was 30.2799 mg/L. In conclusion, recombinant hLyzl6 was expressed successfully by the Pichia pastoris strain GS115 and had in vitro bactericidal activity, which indicated that hLyzl6 may play a role in innate immunity of the male reproductive system and laid a foundation for its functional research and application.
According to the sequences of GP5a gene of XX2012 strain HP-PRRSV, a pair of specificity primers were designed synthesis. The complete gene fragment of GP5a protein was amplified by RT-PCR, and the eukaryotic expression vector pCAGGS-GP5a which containing GP5a protein gene was constructed. Polyclonal antiserums of GP5a protein were prepared by gene immunization pCAGGS-GP5a vector in BALB/c mice. The results showed that the sequences of GP5a protein gene were consistent with predicted size, 156 base pairs. PCR, digestion and sequencing results showed that the recombinant plasmid pCAGGS-GP5a was correct. After the mice were immuned with pCAGGS-GP5a three times, the separated serums of these mice contain the polyclonal antibody of GP5a protein, and the results of indirect immuno-fluorescence assay (IFA) showed that the polyclonal antiserums can specifically recognize cells infected by the virus. In summary, the recombinant plasmid pCAGGS-GP5a containing GP5a protein gene was constructed, and the polyclonal antibody of GP5a protein was prepared, which laid the foundation for the further study of subcellular localization and relevant function of GP5a protein.
According to transcriptome data of Fagopyrum tataricum at flowering, using PCR and RT-PCR techniques, the DNA and cDNA sequences of FtLOL1 (GenBank accession number: KP260662) gene were amplified from Fagopyrum tataricum. The obtained sequences were analyzed by bioinformatics software, and the expression FtLOL1 gene were analysed by qPCR under UV-B, SA and 4℃ cold stress. The results showed that the DNA sequence of FtLOL1 gene was 1 720bp, of which consisted 5 exons and 4 introns, in line with the principle of GT-AG splicing, and the cDNA of FtLOL1 contained a 444bp ORF, encoding 147 amino acids, with the typical features of LSD1 family. The 2mmol/L SA, UV-B radiation and 4℃ cold stress could lead to a significant discrease in the expression of the FtLOL1 gene. The results expected to lay a foundation for the stress resistance study in Fagopyrum tataricum.
The recombinant strains of C. crenatum MT-M4 ΔproC and C. crenatum MT-M4 ΔputP were separately constructed based on genome-scale metabolic network model (GSMN) of Corynebacterium glutamicum. As results shown, L-arginine production of C. crenatum MT-M4 ΔproC was significantly increased by approximately 15.90% higher than that of the original strain, reached to 9.94g/L with glucose transformation rate increased by 26.02%. However, the growth of C. crenatum MT-M4 ΔproC was obviously inhibited. When 24 mmol/L of proline was added, the arginine production reached to 12.22g/L and its growth also got well. The L-arginine production of C. crenatum MT-M4 ΔputP was significantly increased by 42.70% and reached to 12.23g/L with glucose transformation rate increased by 49.31%. The results showed that putP deletion compared with proC deletion was more conducive to L-arginine biosynthesis. The disruption of putP had no influence on the physiological metabolism of the bacteria strain and the mutant strain did not need proline added in medium.
Objective: The purpose is to knock out FgPDE1 gene in Fusarium graminearum, so as to identify the function of the gene through analysis of the phenotype of the deletion mutants. Methods: The Split-marker strategy is applied to build knockout cassette containing hygromycin phosphotransferase gene and anti-hygromycin transformants are obtained by using PEG-mediated protoplast transformation. The FgPDE1 gene deletion mutants are screened by the absent of the PCR products of the FgPDE1 gene. The function of the gene is analyzed according to the mutant phenotype and pathogenicity detection. Results: The Split-marker knockout cassette is successfully constructed and the transformants are obtained after PEG-mediated transformation of the protoplasts of PH-1 and then, three FgPDE1 gene deletion mutants are obtained through PCR screening. The phenotypic analysis revealed that there is no significant difference between ΔFgPDE1 and wild type in terms of colony phenotype and growth rate. The virulence assay by fruit of tomato infected by conidiospores show that the mutants do not decrease greatly in pathogenicity. However, microscopic observation show that the conidiospore amount of ΔFgPDE1 reduce significantly. Conclusions: FgPDE1 gene might be related to conidia formation of the Fusarium graminearum.
Pyridine derivatives are the important value-added chemicals, and biocatalysis is a potential technology for the industrial synthesis of pyridine derivatives. The structure information of 6-hydroxy-3-succinoyl-pyridine monooxygenase (HspB) by mutant construction was investigated. The space structure of HspB has been built through computer modeling and docked with its substrate HSP(2,5-dihydroxy-pyridine). Then 25 mutations of HspB were constructed and studied, according to molecular simulation, sequence alignment, and homologous crystal references. All the mutants have been expressed and purified, and the kinetic parameters of soluble mutants have been measured. According to the properties of mutants, it can infer that the correct binding of FAD possesses important roles in protein stability, and moreover, substrate HSP, and co-enzyme NADH live in the same activity center in HSP but interact with different amino acids. The structure information will help us for the industrial application of pyridine monooxygenase.
Objective: The aim is to screen a strain with high chitinase activity from submarine sediments, to separate chitinase secreted by purified strain, and to analyze its activity by enzymatic methods. Methods: Sediments from Beibu Gulf of South China Sea were chosen as samples. Bacterial strains were screened using the clear zone and DNS method.Chitin binding assay and multiple chromatography methods were used to separate chitinase secreted by purified strain, and studied the enzymatic properties of the purified chitinase. Results: 21 strains of chitinase producers were obtained. Among them, chitinase secreted by Bacillus cereus strain B04 has the highest chitinase activity. A 36 kDa chitinase from the fermentative broth of B. cereus strain B04 was purified. Studies showed that the optimum pH and reaction temperature for the enzyme was pH 4.0 and 60℃, respectively. In pH 3.0~10.0, it has stable activity. Its activity was significantly enhanced by Co2+ and inhibited by Ag+. Conclusion:The basis for the industrial application of chitinase was provided.
Objective:The aims are to obtain thermostable MTG from Streptomyces mobaraensis mutants library producing by nitrosoguanidine (NTG) radom mutagenesis and high-throughput screening.Methods:A rapid method of screening for thermostable MTG from Streptomyces mobaraensis is established by optimization of high-throughput MTG activity assay, screening temperature and time, respectively.Streptomyces mobaraensis mutants library is obtained by optimization of the concentration of NTG. A mutant 12-82 with thermostable MTG was chosen by preliminary screening and second screening from the 5200 mutants library. Results:The spores from Streptomyces mobaraensis were treated by NTG (2mg/ml, pH8.0) for 60 min, then the fermented supernatant was incubated at 70℃ for 7.5 min after fermentation in 96 well microtiters. Finally, MTG activity was determined at 37℃ air bath for 10 min.5 mutants with thermostable MTG were selected from 5200 mutants.When incubated at 50℃ for 60min and 70℃ for 1.5min, the relative activity of 12-82 was almost 20% higher than the parent strain.When incubated at 80℃ for 2min, the relative activity of 12-82 was 11.9%. Conclusion:5 mutants with thermostable MTG were selected by the screening method of NTG random mutagenesis combined with high-throughput screening.Among them, the relative activity of the mutant 12-82 was 10%~20% higher than that of the parent strain when incubated at 50℃,70℃ and 80℃, respectively.This established rapid method to obtain thermostable MTG is practical and the increased thermostability of MTG will be advantageous for its application in food processing at high temperature.
The optimal culture medium of creatininase production on the Arthrobacter nicotiana were carried out. Firstly, using creatine as inductor and metal ions experiment, it was found that creatine and metal ions have a great influence upon the creatininase production. Secondly, single factor experiment showed the optimization carbon and nitrogen sources was soluble starch and corn steep liquor,but compound nitrogen source of yeast extract and corn steep liquor was much better(creatininase production reached 108.5 U/g wet mycelium). At last, creatininase production reached 182.5 U/g wet mycelium under the optimization of fermentation medium, which was about 19.87 times as high as the initial fermentation enzyme activity.
Latent infection in resting memory CD4+ T cells is the major barrier to HIV-1 eradication. Under most circumstances, proviral cDNA was integrated into the host genome. Due to the factors from virus or the host, the transcription was interrupted, without producing any detected viral proteins. So the latently infected cells could not suffer the cytopathic effect from the virus, the attack from cytotoxic T lymphocyte, or the antiviral effect from the anti-retroviral therapy. It is urgent to decrease the latent reservoir in AIDS treatment. In vivo or in vitro latency models are beneficial for further studying the mechanism of latency establishment, maintenance or reactivation. The focuses are mainly on the in vitro HIV-1 latency models using immortalized cell lines, primary resting CD4+ T cells or activated CD4+ T cells.
FGF20 (fibroblast growth factor 20, FGF20) is one of the members of fibroblast growth factor (FGFs) family. It has been found that FGF20 has a wide range of biological activity, not only in brain degenerative neurological diseases such as Parkinson's disease,but also has important biological functions in tissue repair, tumorigenesis, and other aspects of organ development. Although as a recombinant protein possessing drug development potential but its mechanism remains to be further study, yet it has been believed that the biological characteristics of FGF20 has a very broad areas of research and application.
Bioreactor technology can not only break the limitation of environmental conditions, but also contribute to artificial control of the productive process when it is used for growing plant cells in vitro, which creates conditions for large-scale cultivation of plant cells or industrialized production of useful metabolites, and it has been considered as a hot topic in recent work on plant cell culture. Based on the characteristics of plant cell culture, the principles and the advantages and disadvantages of various kinds of bioreactors for plant cells including stirred tank reactor, non-stirred bioreactor, immobilized bioreactor, photo-bioreactor and single-use bioreactor, were compared and analysed. Finally, the future perspectives of bioreactors for plant cell culture were outlined.
Lysozyme, as a powerful natural antibiotic peptide, can be used as an effective alternative to antibiotics and chemical preservatives. With advances of lysozyme, the increasingly severe problems of bacterial resistance, antibiotic residues in dairy products and overdose of chemical preservative which cause food safety issues may be addressed. The research status of lysozyme in terms of muramidase and non-muramidase activity were summarized, and an overview of the research progress in the development of new antimicrobial activity of lysozyme by protein engineering were provided.
The crucial segment in the process of the conversion of lignocellulose into biofuels and high value-added chemicals is the efficient lignocellulose hydrolysis to afford soluble sugars for subsequent fermentation. From the perspective of environmental protection, enzymatic hydrolysis is an effective way to lignocellulose degradation completely without environmental pollution, furthermore, this method has got widely attention because of its advantages, such as lower sugar loss, fewer by-products and mild conditions. Due to the complex compositions and structures of cellulose and some drawbacks of cellulase itself, such as poor stability, short lifetime and low activity, which make low efficiency and high cost of enzymatic saccharification. Scholars from all over the world have carried out researches in many aspects about how to improve the efficiency of lignocellulose enzymatic saccharification.The latest processes of both theoretical and technological research in recent years were summarized. The influences of pretreatment of lignocellulose, cellulase production strains and technology, cellulase complex and recombination, cellulase additives, cellulase immobilization, external fields, recycling and reuse of cellulase were reviewed, and the prospects on lignocellulose enzymatic saccharification in the future were also presented.
