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1.
Chinese Journal of Biotechnology ; (12): 2956-2966, 2021.
Article in Chinese | WPRIM | ID: wpr-887857

ABSTRACT

Teaching in experiments of biology is important for the cultivation of life science talents. In view of the rapid development of life science and the increasing demand for research-oriented talent training, teaching in experiments of biology should set up a variety of learning outcomes: to train experimental skill, to cultivate students' experimental design and operation abilities, and to improve students' scientific thinking and innovative consciousness. We have carried out an educational reform on experimental genetic engineering blended course. In this paper, we introduced our methods of organizing online materials, the curriculum design of the blended course, the implementation details, and a preliminary analysis of teaching effects. We found that experimental genetic engineering blended course could support students' active learning and a learning-centered teaching model. Moreover, it could facilitate students' achievement of improving experimental skills, cultivating a rigorous scientific attitude, professional research quality and academic innovation ability.


Subject(s)
Biological Science Disciplines , Curriculum , Genetic Engineering , Humans , Students
2.
Chinese Journal of Biotechnology ; (12): 2116-2126, 2021.
Article in Chinese | WPRIM | ID: wpr-887785

ABSTRACT

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.


Subject(s)
CRISPR-Cas Systems/genetics , Genetic Engineering , Ribosomes , Spiramycin , Streptomyces/genetics
3.
Chinese Journal of Biotechnology ; (12): 1457-1463, 2021.
Article in Chinese | WPRIM | ID: wpr-878648

ABSTRACT

Starting from participating the high-level professional competition, our school has built a talent training system with the spirit of "biomaker" and an innovative practical ability training system. Such system takes the interest of student as the starting point, and relies on the strong scientific research and teaching infrastructure. The programme gives full play to students' initiatives and enhances the scientific research literacy and comprehensive ability of undergraduates majoring in biotechnology. It is an effective exploration of the traditional university education model and meets the urgent demand for innovative talents training in the era of rapid development of life sciences.


Subject(s)
Biological Science Disciplines , Biotechnology , Genetic Engineering , Humans , Students , Universities
4.
Gac. méd. Méx ; 156(1): 53-59, ene.-feb. 2020.
Article in Spanish | LILACS | ID: biblio-1249870

ABSTRACT

Resumen En este ensayo se analizan las implicaciones bioéticas de la reciente manipulación genética en embriones humanos con CRISPR-Cas9 para eliminar el gen CCR5 y el nacimiento de dos gemelas discordantes. El experimento se divulgó en medios sociales. Los principales problemas bioéticos identificados son la justificación del modelo, el proceso de consentimiento informado y la falta de declaración de evidentes conflictos de interés. No se evaluaron apropiadamente las consecuencias del experimento sobre la vida de las gemelas nacidas como la afectación a su autonomía, los supuestos beneficios por recibir y los riesgos futuros de daño durante su vida. Habiendo manipulado la línea celular germinal, no se consideraron los efectos sobre su descendencia futura. Este tipo de acciones tiene un impacto negativo en la forma como la sociedad concibe la ciencia. La ingeniería genética debe reservarse al contexto experimental básico o bien como investigación cínica para la corrección de enfermedades conocidas graves de origen genético, bajo estricta supervisión regulatoria y bioética y de manera gradualista de acuerdo con el progreso de las técnicas de edición genética.


Abstract In this essay, the bioethical implications of the recent genetic manipulation in human embryos with CRISPR-Cas9 to eliminate the CCR5 gene and the birth of a pair of discordant twin girls are analyzed. The experiment was disseminated via social media. The main bioethical flaws identified include the justification of the model, the informed consent process and the lack of disclosure of evident conflicts of interest. The consequences of the experiment on the life of the twins that were born were not properly evaluated, such as the impact on their autonomy, the alleged benefits to be received and the future risks of harm during their lifetime. Having manipulated the germ cell line, the effects on their future offspring were not considered. This type of actions negatively affects the way society conceives science. Genetic engineering should be reserved to the basic experimental context or as clinical research for the correction of known serious diseases of genetic origin under strict regulatory and bioethical supervision and using a gradualist approach in accordance with the advances of gene editing techniques.


Subject(s)
Humans , Female , Receptors, CCR5/genetics , CRISPR-Cas Systems , Gene Editing/ethics , Publishing/ethics , Research Design , Twins, Dizygotic , Genetic Engineering/classification , Genetic Engineering/ethics , Genome, Human , HIV Infections/prevention & control , China , Conflict of Interest , Sperm Injections, Intracytoplasmic , Bioethical Issues , Therapeutic Human Experimentation/ethics , Informed Consent/ethics
5.
Blood Research ; : 10-16, 2020.
Article in English | WPRIM | ID: wpr-820808

ABSTRACT

Acute myeloblastic leukemia (AML) is the most frequent acute leukemia in adulthood with very poor overall survival rates. In the past few decades, significant progresses had led to the findings of new therapeutic approaches and the better understanding of the molecular complexity of this hematologic malignancy. Leukemic stem cells (LSCs) play a key role in the initiation, progression, regression, and drug resistance of different types of leukemia. The cellular and molecular characteristics of LSCs and their mechanism in the development of leukemia had not yet been specified. Therefore, determining their cellular and molecular characteristics and creating new approaches for targeted therapy of LSCs is crucial for the future of leukemia research. For this reason, the recognition of surface maker targets on the cell surface of LSCs has attracted much attention. CD33 has been detected on blasts in most AML patients, making them an interesting target for AML therapy. Genetic engineering of T cells with chimeric antigen receptor (CAR-T cell therapy) is a novel therapeutic strategy. It extends the range of antigens available for use in adoptive T-cell immunotherapy. This review will focus on CAR-T cell approaches as well as monoclonal antibody (mAB)-based therapy, the two antibody-based therapies utilized in AML treatment.


Subject(s)
Drug Resistance , Genetic Engineering , Hematologic Neoplasms , Humans , Immunotherapy , Leukemia , Leukemia, Myeloid, Acute , Neoplastic Stem Cells , Receptors, Antigen , Stem Cells , Survival Rate , T-Lymphocytes
6.
Chinese Journal of Biotechnology ; (12): 678-692, 2020.
Article in Chinese | WPRIM | ID: wpr-827001

ABSTRACT

As water-soluble, natural pigments, anthocyanins are responsible for the red, purple and blue colors of many flowers, which attract pollinators to spread pollen. The colors of flowers are also essential for plants to survive in the nature and become one of the most significant characteristics of ornamental plants. In the booming floriculture industry, to produce various flower colors could increase the richness of natural colors, but it is still difficult to breed flowers with coveted blue color. The diversity of flower color is mainly determined by the types and contents of anthocyanins and their derivatives. The synthesis of delphinidin pigments is the key factor for breeding blue flowers. However, there are no structural genes in many plants to biosynthesize delphinidin pigments. Blue flowers are successfully created by genetic engineering in recent years. In this paper, using common ornamental plants as examples, we review the mechanism of plant flower coloration from the aspects of the key factors affecting the synthesis of delphinidin pigment and the production strategies of blue flowers based on the regulation of anthocyanin metabolism. Different strategies of molecular breeding could provide opportunities to improve colors of other floriculture plants and to develop anthocyanin-rich economic crops, such as colored cotton with blue fibers.


Subject(s)
Anthocyanins , Metabolism , Flowers , Gene Expression Regulation, Plant , Genetic Engineering , Pigmentation , Genetics
7.
Chinese Journal of Biotechnology ; (12): 891-898, 2020.
Article in Chinese | WPRIM | ID: wpr-826887

ABSTRACT

Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.


Subject(s)
Amino Acids , Genetics , Amino Acyl-tRNA Synthetases , Genetic Engineering , Protein Engineering , RNA, Transfer , Viral Vaccines
8.
Chinese Journal of Biotechnology ; (12): 1269-1276, 2020.
Article in Chinese | WPRIM | ID: wpr-826850

ABSTRACT

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.


Subject(s)
Adenovirus Infections, Human , Epidemiology , Virology , Adenoviruses, Human , Genetics , Genetic Engineering , Methods , Genetic Vectors , Humans , Oncolytic Virotherapy , Oncolytic Viruses , Genetics , Virus Replication
9.
Chinese Journal of Biotechnology ; (12): 2193-2205, 2020.
Article in Chinese | WPRIM | ID: wpr-878478

ABSTRACT

Endoglucanase (EG) is an important component of cellulases and play an important role in cellulose degradation. However, its application is limited due to the low yield of endoglucanase from natural microorganisms. Efficient heterologous expression of endoglucanase is an effective way to solve this problem. To obtain the engineered Saccharomyces cerevisiae for high-yield endoglucanase, endoglucanase gene was cloned from Clostridium cellulovorans, with a total length of 1 996 bp, encoding 440 amino acids, and the complete expression cassette (PαEGC) was constructed with the PGK promoter sequence from Saccharomyces cerevisiae, α-signal peptide sequence from pPIC9K plasmid and CYC1 terminator sequence from pSH65 plasmid by gene splicing by overlap extension PCR (SOE PCR), and the expression vector of endoglucanase in Saccharomyces cerevisiae was constructed by rDNA integration. The relationship between copy number and protein expression was explored. Random multicopy expression of endoglucanase was performed in Saccharomyces cerevisiae. The copy number of endoglucanase was identified by Droplet Digital PCR and explore the relationship between copy number and protein expression.The engineered Saccharomyces cerevisiae of endoglucanase with copy numbers of 1, 3, 4, 7, 9, 11, 15, 16, 19, 21, 22 and 23 were obtained by rDNA integration, respectively. The results showed that when the copy number was 15, the enzyme activity was the highest, namely 351 U/mL. The engineered strain of Saccharomyces cerevisiae for endoglucanase was successfully constructed, which can provide reference for the heterologous expression of other industrial enzymes.


Subject(s)
Cellulase/genetics , Genetic Engineering , Industrial Microbiology , Plasmids/genetics , Saccharomyces cerevisiae/genetics
10.
Chinese Journal of Biotechnology ; (12): 2162-2170, 2020.
Article in Chinese | WPRIM | ID: wpr-878475

ABSTRACT

We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.


Subject(s)
4-1BB Ligand/metabolism , Cell Line, Tumor , Genetic Engineering , Humans , Inducible T-Cell Co-Stimulator Protein/metabolism , Multiple Myeloma/therapy , Signal Transduction , T-Lymphocytes/chemistry , Xenograft Model Antitumor Assays
11.
S. Afr. med. j. (Online) ; 109(8): 54-58, 2019. ilus
Article in English | AIM, AIM | ID: biblio-1271230

ABSTRACT

South Africa encompasses extraordinary genetic diversity, frequently revealing unique mutations and variations associated with disease. Despite the advances of traditional gene therapy, our understanding of causative mutations in the South African population has, for the most part, contributed to diagnostic rather than therapeutic interventions. Recent developments in genome engineering and its ease of use have released a powerful tool with which to intervene in otherwise untreatable disease. In addition, harnessing this tool for discrete genetic edits provides a mechanism by which screening of new drugs specific to our population's diversity can be accomplished. Here, we use examples of some of the most advanced genome engineering approaches to develop therapeutic strategies that would specifically affect South African individuals


Subject(s)
Genetic Engineering , Genome , South Africa , Therapeutics
13.
Braz. j. med. biol. res ; 52(5): e8108, 2019. tab
Article in English | LILACS | ID: biblio-1001521

ABSTRACT

Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.


Subject(s)
Humans , Animals , Male , Female , Rabbits , Animals, Genetically Modified , Genetic Engineering/methods , Disease Models, Animal , Atherosclerosis/physiopathology , Transcription Activator-Like Effector Nucleases/metabolism , Gene Transfer Techniques , Biomedical Research/methods , Atherosclerosis/genetics
14.
Chinese Journal of Biotechnology ; (12): 956-971, 2019.
Article in Chinese | WPRIM | ID: wpr-771831

ABSTRACT

Silk-based biomaterials are featured with excellent mechanical properties, good biocompatibility and biodegradability, which contribute to their potential applications in biomedical field. The current recognition of silk protein materials in structure and function provides a basic theory for the transformation of silk protein into new types of biomaterials. In addition, exogenous sequences encoding new peptide or structural domain can be inserted into the maternal gene sequences encoding silk proteins through genetic engineering technology to synthesize novel silk-based biomaterials with unique functions. This review summarizes the current trend and development perspective of genetically engineered functional silk-based materials for biomedical applications.


Subject(s)
Biocompatible Materials , Genetic Engineering , Hydrogels , Silk
15.
Chinese Journal of Biotechnology ; (12): 1662-1675, 2019.
Article in Chinese | WPRIM | ID: wpr-771764

ABSTRACT

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.


Subject(s)
Bacterial Proteins , Genetic Engineering , Insecticides , Macrolides , Saccharopolyspora
16.
Chinese Journal of Biotechnology ; (12): 1761-1770, 2019.
Article in Chinese | WPRIM | ID: wpr-771755

ABSTRACT

Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.


Subject(s)
Escherichia coli , Escherichia coli Proteins , Genetic Engineering , Plasmids , Recombination, Genetic
17.
Chinese Journal of Biotechnology ; (12): 472-481, 2019.
Article in Chinese | WPRIM | ID: wpr-771360

ABSTRACT

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Genetic Engineering , Homologous Recombination
18.
Frontiers of Medicine ; (4): 69-82, 2019.
Article in English | WPRIM | ID: wpr-771259

ABSTRACT

Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.


Subject(s)
Cell Line, Tumor , Cytokines , Metabolism , Cytotoxicity, Immunologic , Genetic Engineering , HLA-A2 Antigen , Metabolism , Humans , Immunotherapy, Adoptive , Methods , Lymphocyte Activation , Receptors, Antigen, T-Cell , Genetics , Allergy and Immunology , T-Lymphocytes , Allergy and Immunology
19.
Article in English | WPRIM | ID: wpr-758930

ABSTRACT

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21–42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-γ and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.


Subject(s)
Adenoviridae , Adenovirus Vaccines , Antibodies , Antibodies, Neutralizing , Capsid , Capsid Proteins , Circovirus , Genetic Engineering , Immunization , Interleukin-13 , Lung , Lymph Nodes , Lymphocytes , Swine , Yersinia pseudotuberculosis
20.
Vaccimonitor (La Habana, Print) ; 27(2)mayo.-ago. 2018. ilus, tab
Article in Spanish | LILACS, CUMED | ID: biblio-1094605

ABSTRACT

Para las metodologías de ADN recombinante, los biólogos moleculares emplean mutantes de Escherichia coli que son adquiridos de kit comerciales o de colecciones microbianas especializadas. En la práctica estos mutantes son conservados por pases sucesivos o en un banco poco caracterizado. No seguir el sistema de lotes de siembra (lote de siembra de referencia y lotes de siembra de trabajo) y la falta de controles sistemáticos, puede llevar a la pérdida de las características originales de las cepas y afectar la calidad de los resultados experimentales. Por otra parte, la propia dinámica de los laboratorios de investigación hace que sea poco práctico realizar las extensas verificaciones propias del trabajo de las colecciones microbianas. El objetivo de este artículo es proponer un método de evaluación de pureza y estabilidad genética que permita la verificación de varios mutantes de E. coli en un solo ensayo. En él se definen los criterios de selección para el diseño de medios de cultivos específicos. Se realizan diluciones seriadas de los cultivos crecidos en medio Luria Bertani y se emplea como método de siembra las trazas de dilución. Los resultados evidencian que teniendo en cuenta las rutas metabólicas afectadas en cada mutante, se pueden agrupar varias cepas a verificar en un solo ensayo. La combinación de medios específicos permite tener un criterio de la pureza del cultivo y la estabilidad genética. Esta alternativa permite el chequeo rápido de aquellos marcadores de las cepas que son determinantes en las metodologías de ADN recombinante(AU)


For recombinant DNA methodologies, molecular biologists use Escherichia coli mutants acquired from commercial kits or specialized microbial strain collections. These mutants are routinely conserved by successive passages or in poorly-characterized strain banks. A failure to follow the seed lot system (reference seed lot and working seed lot) and the lack of systematic controls, can lead to the loss of original strain characteristics and affect the quality of experimental results. On the other hand, the dynamic of research laboratories makes impractical to carry out extensive verifications related to the work with microbial collections. The aim of this article is to propose a single-assay method for genetic purity and stability evaluation of multiple E. coli mutants simultaneously. For the design of specific culture media, selection criteria were defined. Serial dilutions of cultures in Luria Bertani medium were made, and track dilution was used as seeding method. The results show how a mutated metabolic pathway-oriented design permit the verification of several strains in a single trial. A criterion about culture purity and genetic stability could be obtained after combining specific media. This alternative allows for a rapid evaluation of strain key genetic markers for recombinant DNA methodologies(AU)


Subject(s)
DNA, Recombinant , Genetic Engineering , Escherichia coli , Genotype
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