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1.
Int J Biol Macromol ; 160: 736-740, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32485251

RESUMO

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic in the past four months and causes respiratory disease in humans of almost all ages. Although several drugs have been announced to be partially effective treatments for this disease, no approved vaccine is available. Here, we described the construction of a recombinant Lactobacillus plantarum strain expressing the SARS-CoV-2 spike protein. The results showed that the spike gene with optimized codons could be efficiently expressed on the surface of recombinant L. plantarum and exhibited high antigenicity. The highest protein yield was obtained under the following conditions: cells were induced with 50 ng/mL SppIP at 37 °C for 6-10 h. The recombinant spike (S) protein was stable under normal conditions and at 50 °C, pH = 1.5, or a high salt concentration. Recombinant L. plantarum may provide a promising food-grade oral vaccine candidate against SARS-CoV-2 infection.


Assuntos
DNA Recombinante/genética , Engenharia Genética/métodos , Lactobacillus plantarum/genética , Glicoproteína da Espícula de Coronavírus/genética , Expressão Gênica
2.
PLoS One ; 15(4): e0231886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320461

RESUMO

Cotton leaf curl disease (CLCuD), caused by whitefly (Bemisiatabaci) transmitted single-stranded DNA viruses belonging to the Genus, Begomovirus (family, Geminiviridae) in association with satellite molecules; is responsible for major economic losses in cotton in three northwest (NW) Indian states Haryana, Punjab, and Rajasthan. Annual CLCuD incidences during 2012 to 2014 were estimated to be 37.5%, 63.6%, and 38.8% respectively. Cotton leaves were collected from symptomatic plants annually for three years and subjected to DNA isolation, followed by rolling circle amplification (RCA), cloning, and DNA sequencing of apparently full-length begomoviral genomes and associated betasatellites and alphasatellites. Among the thirteen CLCuD-begomoviral genomes recovered, eight were identified as Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra), one as -Pakistan (PK) and another as -Faisalabad (Fai), whereas, three were as Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu), indicating that CLCuMuV-Ra was the most prevalent begomovirus species. Five of the eight CLCuMuV-Ra sequences were found to be recombinants. The CLCuMuV-Ra- associated satellites consisted of Cotton leaf curl Multan betasatellite (CLCuMB), and Gossypium darwinii symptomless alphasatellite (GDarSLA), and Croton yellow vein mosaic alphasatellite (CrYVMoA). The second most abundant helper virus species, CLCuKoV-Bu, was associated with CLCuMB and GDarSLA.


Assuntos
DNA Recombinante/genética , Surtos de Doenças , Gossypium/virologia , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Evolução Molecular , Índia
3.
DNA Cell Biol ; 39(6): 992-999, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32326732

RESUMO

Peste des petits ruminants (PPR) is an acute, highly infectious, and highly pathogenic disease, which mainly damages small ruminants such as goats and sheep. Hemagglutinin protein (H), the main antigenic protein of peste des petits ruminants virus (PPRV), has been a hot spot in the research of genetic engineering vaccine for PPRV. In this study, the silkworm baculovirus surface display technology is combined with the transmembrane structure of the silkworm baculovirus envelope protein GP64 and different characteristics of the promoters to display four kinds of fusion proteins, which contain Pph-H, Pph-HJ, Pie1-H, and Pie1-HJ. The fusion proteins displayed on baculovirus surface have been detected by western blotting, cell surface immunofluorescence, and immunogold electron microscopy. In addition, the dominant form of PPR H displayed on baculovirus surface has been determined which is fusion protein mediated by Pph containing the hemagglutinin protein and full-length GP64, Pph-H. Furthermore, by comparing the fluorescence intensity of binding of hemagglutinin protein and signaling lymphocyte activation molecules (SLAM) in Vero-SLAM cells by immunocytochemistry, Pph-H can be combined with the receptor protein of PPRV, SLAM. It provides technical support for displaying the different structure of hemagglutinin and exploring the key sites of hemagglutinin and SLAM binding. Meanwhile, it is important for exploring the pathogenesis and immune mechanism of PPRV.


Assuntos
Baculoviridae/metabolismo , Hemaglutininas/metabolismo , Interações entre Hospedeiro e Microrganismos , Vírus da Peste dos Pequenos Ruminantes/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Animais , Baculoviridae/genética , Bombyx/virologia , Chlorocebus aethiops , DNA Recombinante/genética , Ligação Proteica , Células Vero
4.
Virology ; 542: 20-27, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31957662

RESUMO

Tomato yellow leaf curl virus (TYLCV) and its related viruses are prone to recombination. It was reported that random homologous recombination between 20% diverging TYLCV related species is rarely deleterious and may be associated with a fitness advantage. Indeed, TYLCV-IS76, a recombinant between the 20% divergent TYLCV and tomato yellow leaf curl Sardinia virus (TYLCSV), exhibited a higher fitness than that of parental viruses. As this typical fitness advantage was observed with TYLCV-IS76 representatives of different pedigrees, it was thought that it is induced by beneficial intra-genomic interactions rather than by specific mutations. This hypothesis was further supported with TYLCV-IS141, a TYLCV recombinant with a short TYLCSV inherited fragment of around 141 nts, slightly longer than that of TYLCV-IS76. Indeed, the typical fitness advantage was detected irrespective of the position of the recombination breakpoint (loci 76 or 141) and the sequences of the TYLCV and TYLCSV inherited fragments.


Assuntos
Begomovirus/genética , Lycopersicon esculentum/virologia , Begomovirus/patogenicidade , Begomovirus/fisiologia , DNA Recombinante/genética , DNA Recombinante/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Resistência à Doença/genética , Aptidão Genética , Genoma Viral , Lycopersicon esculentum/genética , Mutagênese Sítio-Dirigida , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/virologia , Recombinação Genética , Especificidade da Espécie
5.
Artif Cells Nanomed Biotechnol ; 48(1): 259-265, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31851845

RESUMO

A metal-resistant engineered Pichia pastoris was developed here to fulfil the metal bioleaching in aqueous conditions. Parent and recombinant yeasts were grown in YPD medium containing different concentrations of ion metals. XRD, electron microscopy and particle size analyser were used for the characterisation and the nanoparticle analyses. The nanoparticle production kinetics were studied by ICP-OES. The cytotoxicity of nanoparticles was assayed against human cell lines. Media colours changed to a range from purplish-brown to grey during early fermentation stages. The maximum biosorption capacities were recorded 81.23 and 493.35 mg/g for gold and palladium in batch conditions, respectively. Various physical investigations proved monodispersed spherical nanoparticles around 100 nm in size. Pure palladium nanoparticles and PdCl2 represented the least cytotoxic potency towards T47D and EPG85.257 cells. The results demonstrated that the genetically modified yeast is a cost-effective, high-throughput, robust, and facile system for metal biosorption.


Assuntos
Ouro/química , Ouro/metabolismo , Nanopartículas Metálicas , Paládio/química , Paládio/metabolismo , Pichia/genética , Pichia/metabolismo , Biotecnologia , Linhagem Celular , Cor , DNA Recombinante/genética , Ouro/toxicidade , Cinética , Organismos Geneticamente Modificados , Paládio/toxicidade , Pichia/crescimento & desenvolvimento
6.
Essays Biochem ; 63(4): 457-468, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31652313

RESUMO

DNA present in all our cells acts as a template by which cells are built. The human genome project, reading the code of the DNA within our cells, completed in 2003, is undoubtedly one of the great achievements of modern bioscience. Our ability to achieve this and to further understand and manipulate DNA has been tightly linked to our understanding of the bacterial and viral world. Outside of the science, the ability to understand and manipulate this code has far-reaching implications for society. In this article, we explore some of the basic techniques that enable us to read, copy and manipulate DNA sequences alongside a brief consideration of some of the implications for society.


Assuntos
DNA Recombinante/genética , DNA/genética , Testes Genéticos , Plantas Geneticamente Modificadas/genética , Clonagem Molecular/métodos , DNA/isolamento & purificação , Eletroforese em Gel de Ágar/métodos , Vetores Genéticos/genética , Mutação , Reação em Cadeia da Polimerase/métodos
7.
J Neonatal Perinatal Med ; 12(3): 333-338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31476172

RESUMO

 We report a case of two consecutive pregnancies in the same couple presenting with very low pregnancy-associated plasma protein A (PAPP-A), with both pregnancies affected by multiple anomalies of a similar phenotype identified during mid-trimester ultrasound, and eventual diagnosis of Peters-plus syndrome. This case is important in expanding the differential for very low PAPP-A. It also demonstrates the diagnostic value of whole-exome sequencing (WES) after prenatal diagnosis of recurrent fetal ultrasonographic findings. The importance and complexity of providing patient education to enable informed consent for next generation sequencing technologies is discussed.


Assuntos
Anormalidades Múltiplas/genética , Fenda Labial/diagnóstico , Córnea/anormalidades , Transtornos do Crescimento/diagnóstico , Deformidades Congênitas dos Membros/diagnóstico , Proteína Plasmática A Associada à Gravidez/deficiência , Sequenciamento Completo do Exoma , Anormalidades Múltiplas/diagnóstico , Adulto , Biomarcadores/metabolismo , Fenda Labial/genética , DNA Recombinante/genética , Feminino , Transtornos do Crescimento/genética , Humanos , Deformidades Congênitas dos Membros/genética , Imagem por Ressonância Magnética , Mutação/genética , Gravidez , Resultado da Gravidez , Proteína Plasmática A Associada à Gravidez/genética , Diagnóstico Pré-Natal , Recidiva
8.
Int J Biol Macromol ; 141: 1287-1292, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499107

RESUMO

ß-conglycinin is one of the most allergenic proteins, and its constituent subunits α', α, and ß are all potential allergens to humans. In the present study, we concentrated on the destructed antigenic sites of ß subunit of ß-conglycinin after high hydrostatic pressure (HHP) treatment. In this paper, the overlapping gene fragments of the ß subunit of ß-conglycinin were amplified by polymerase chain reaction (PCR) and cloned into T7 phage vectors. After being packaged in vitro, the recombinant T7 phage was constructed, and the overlapping fragments of the ß subunit were displayed on the phage surface. The recombinant phages that expressed the overlapping fragments of the ß subunit were used to react with specific antiserum by indirect ELISA to identify the HHP destructed antigenic sites. After three rounds of expression and identification, we used synthetic peptide technology to identify that the obtained fragment was a conformational epitope. We further confirmed that HHP treatment changed the conformational structure of ß-conglycinin, which reduced the antigenicity of the protein.


Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Epitopos/genética , Engenharia Genética , Globulinas/genética , Globulinas/imunologia , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/imunologia , Proteínas de Soja/genética , Proteínas de Soja/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/química , Bacteriófagos/genética , DNA Recombinante/genética , Globulinas/química , Pressão Hidrostática , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Proteínas de Armazenamento de Sementes/química , Proteínas de Soja/química
9.
Lett Appl Microbiol ; 69(5): 366-372, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31508837

RESUMO

We evaluate the efficacy of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia infection through intranasal administration route by targeting the mucosal immunity. The DNA vaccine was constructed and subjected to animal study using the Sprague Dawley (SD) rat. The study was divided into two major parts: (i) active and (ii) passive immunization studies, involving 30 animals for each part. Each group was then divided into five test groups: two test samples G1 and G2 with 50 and 100 µg ml-1 purified DNA vaccine; one positive control G5 with 106  CFU per ml formalin-killed PMB2; and two negative controls, G3 and G4 with normal saline and pVAX1 vector. Both studies were conducted for the determination of immunogenicity by total white blood cell count (TWBC), indirect ELISA and histopathological changes for the presence of the bronchus-associated lymphoid tissue (BALT). Our findings demonstrate that TWBC, IgA and IgG increased after each of the three vaccination regimes: groups G1, G2 and G5. Test samples G1 and G2 showed significant differences (P < 0·05) compared to the negative controls, G3 and G4, but no significant differences from the positive control G5. Groups G1, G2 and G5 showed more formation of BALT compared to the negative controls, G3 and G4. Our results show that intranasal inoculation of recombinant DNA vaccine ABA392 can provoke mucosal immunity which makes it a potential prophylactic against HS. SIGNIFICANCE AND IMPACT OF THE STUDY: New approach of combating haemorrhagic septicaemia disease among bovines by recombinant DNA vaccine is crucial to overcome the loss of edible products from the infected bovines. DNA vaccine can potentially serve as a better immunogen which would elicit both cellular and humoral immunity, and it is also stable for its molecular reproduction. This research report demonstrates an effective yet simple way of administering the DNA vaccine via the intranasal route in rats, to provoke the mucosal immunity through the development of immunoglobulins IgA, IgG and bronchus-associated lymphoid tissue which guard as the first-line defence at the host's mucosal lining.


Assuntos
Vacinas Bacterianas/administração & dosagem , Doenças dos Bovinos/prevenção & controle , Septicemia Hemorrágica/veterinária , Pasteurella multocida/imunologia , Vacinas de DNA/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , DNA Recombinante/imunologia , Ensaio de Imunoadsorção Enzimática , Septicemia Hemorrágica/imunologia , Septicemia Hemorrágica/microbiologia , Septicemia Hemorrágica/prevenção & controle , Imunização Passiva , Masculino , Pasteurella multocida/genética , Ratos , Ratos Sprague-Dawley , Vacinas de DNA/genética , Vacinas de DNA/imunologia
10.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31427451

RESUMO

Nontypeable Haemophilus influenzae (NTHi) is the primary cause of bacterially induced acute exacerbations of chronic obstructive pulmonary disease (COPD). NTHi adheres to and invades host respiratory epithelial cells as a means to persist in the lower airways of adults with COPD. Therefore, we mined the genomes of NTHi strains isolated from the airways of adults with COPD to identify novel proteins to investigate their role in adherence and invasion of human respiratory epithelial cells. An isogenic knockout mutant of the open reading frame NTHI1441 showed a 76.6% ± 5.5% reduction in invasion of human bronchial and alveolar epithelial cells at 1, 3, and 6 h postinfection. Decreased invasion of the NTHI1441 mutant was independent of either intracellular survival or adherence to cells. NTHI1441 is conserved among NTHi genomes. Results of whole-bacterial-cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry experiments identified that NTHI1441 has epitopes expressed on the bacterial cell surface. Adults with COPD develop increased serum IgG against NTHI1441 after experiencing an exacerbation with NTHi. This study reveals NTHI1441 as a novel NTHi virulence factor expressed during infection of the COPD lower airways that contributes to invasion of host respiratory epithelial cells. The role in host cell invasion, conservation among strains, and expression of surface-exposed epitopes suggest that NTHI1441 is a potential target for preventative and therapeutic interventions for disease caused by NTHi.


Assuntos
Células Epiteliais/microbiologia , Haemophilus influenzae/fisiologia , Mucosa Respiratória/citologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , DNA Bacteriano , DNA Recombinante/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Infecções por Haemophilus/microbiologia , Humanos , Doença Pulmonar Obstrutiva Crônica/microbiologia
11.
PLoS One ; 14(8): e0221164, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31454364

RESUMO

Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.


Assuntos
DNA Recombinante/genética , Técnicas de Introdução de Genes/métodos , Marcação de Genes/métodos , Vetores Genéticos/genética , Animais , Callithrix/genética , Clonagem Molecular/métodos , Desoxirribonucleases/genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Humanos , Pesquisa com Células-Tronco
12.
Artif Cells Nanomed Biotechnol ; 47(1): 2593-2604, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31240960

RESUMO

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Quitosana/química , Portadores de Fármacos/química , Escherichia coli O157/imunologia , Imunização , Nanopartículas/química , Animais , Anticorpos Antibacterianos/imunologia , Células 3T3 BALB , Aderência Bacteriana , Chlorocebus aethiops , DNA Recombinante/genética , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/fisiologia , Feminino , Camundongos , Células Vero
13.
Appl Biochem Biotechnol ; 189(3): 1007-1019, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31161382

RESUMO

A heterologous xylose utilization pathway, either xylose reductase-xylitol dehydrogenase (XR-XDH) or xylose isomerase (XI), is usually introduced into Saccharomyces cerevisiae to construct a xylose-fermenting strain for lignocellulosic ethanol production. To investigate the molecular basis underlying the effect of different xylose utilization pathways on the xylose metabolism and ethanol fermentation, transcriptomes of flocculating industrial strains with the same genetic background harboring different xylose utilization pathways were studied. A different source of xylA did not obviously affect the change of the strains transcriptome, but compared with the XR-XDH strain, several key genes in the central carbon pathway were downregulated in the XI strains, suggesting a lower carbon flow to ethanol. The carbon starvation caused by lower xylose metabolism in XI strains further influenced the stress response and cell metabolism of amino acid, nucleobase, and vitamin. Besides, the downregulated genes mostly included those involved in mitotic cell cycle and the cell division-related process. Moreover, the transcriptomes analysis indicated that the after integrate xylA in the δ region, the DNA and chromosome stability and cell wall integrity of the strains were affected to some extent. The aim of this was to provide some reference for constructing efficient xylose-fermenting strains.


Assuntos
Perfilação da Expressão Gênica , Indústrias , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , DNA Recombinante/genética , Fermentação , Fatores de Transcrição/metabolismo
14.
Circulation ; 140(7): 566-579, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31177839

RESUMO

BACKGROUND: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. METHODS: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. RESULTS: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. CONCLUSIONS: Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.


Assuntos
Proliferação de Células/fisiologia , DNA Recombinante/biossíntese , Miócitos Cardíacos/metabolismo , Serina-Treonina Quinases TOR/biossíntese , Proteína 1 de Ligação a X-Box/biossíntese , Adolescente , Adulto , Animais , Animais Recém-Nascidos , Células Cultivadas , DNA Recombinante/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/genética , Proteína 1 de Ligação a X-Box/genética , Adulto Jovem
15.
Mol Biotechnol ; 61(8): 579-601, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31168761

RESUMO

Microbes are ubiquitously distributed in nature and are a critical part of the holobiont fitness. They are perceived as the most potential biochemical reservoir of inordinately diverse and multi-functional enzymes. The robust nature of the microbial enzymes with thermostability, pH stability and multi-functionality make them potential candidates for the efficient biotechnological processes under diverse physio-chemical conditions. The need for sustainable solutions to various environmental challenges has further surged the demand for industrial enzymes. Fueled by the recent advent of recombinant DNA technology, genetic engineering, and high-throughput sequencing and omics techniques, numerous microbial enzymes have been developed and further exploited for various industrial and therapeutic applications. Most of the hydrolytic enzymes (protease being the dominant hydrolytic enzyme) have broad range of industrial uses such as food and feed processing, polymer synthesis, production of pharmaceuticals, manufactures of detergents, paper and textiles, and bio-fuel refinery. In this review article, after a short overview of microbial enzymes, an approach has been made to highlight and discuss their potential relevance in biotechnological applications and industrial bio-processes, significant biochemical characteristics of the microbial enzymes, and various tools that are revitalizing the novel enzymes discovery.


Assuntos
Proteínas de Bactérias , Enzimas , Proteínas Fúngicas , Microbiologia Industrial , Engenharia Metabólica , DNA Recombinante/genética , DNA Recombinante/metabolismo
16.
J Biotechnol ; 301: 18-23, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31158410

RESUMO

Many recent epigenetic studies utilize the advantages of CRISPR/dCas9 based tools in linking certain epigenetic modification with gene expression regulation. Various multifactorial diseases often contain changed epigenetic signatures at many loci, so tools for simultaneously targeting different loci would significantly facilitate the understanding of disease pathogenesis. We tested different dCas9 orthologs (dCjCas9, dNmCas9, dSt1Cas9, dFnCas9, dSaCas9 and dSpCas9) in C-terminal fusion with DNMT3A effector domain to find candidates that potentiate effector domain to perform its function at the target site. We demonstrated that nuclear localization signals (NLS) at both termini of fusion constructs is crucial for both proper nuclear import of such large constructs as well as for maximization of targeted DNA methylation activity. We identified SpCas9, SaCas9 and CjCas9 as potential candidates for the fusion constructs. With further optimization of the SaCas9 ortholog, due to less complex PAM requirements in contrast to CjCas9, we showed that N-terminal fusion with DNMT3A (dSaCas9-DNMT3A) is optimal to exert targeted DNA methylation activity comparable to the dSpCas9-DNMT3A construct. N-terminal fusions showed better results for both Cas9 orthologs, SaCas9 and SpCas9, so it can be used as universal approach for linking different effector domains in order to obtain highly active fusions.


Assuntos
Sistemas CRISPR-Cas/genética , DNA Recombinante/genética , Edição de Genes/métodos , Fusão Gênica/genética , Epigenômica/métodos , Regulação da Expressão Gênica
17.
JAMA Netw Open ; 2(6): e195752, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31199449

RESUMO

Importance: The PROM1 gene, commonly associated with cone-rod dystrophies, may have dominant or recessive phenotypes that influence disease onset and severity. Objective: To characterize the clinical phenotype and molecular genetic variations in patients with PROM1 variants. Design, Setting, and Participants: This case-series study was conducted at 2 specialist retinal genetics clinics and examined 19 consecutively enrolled patients with PROM1-related retinal degeneration. Data were collected and analyzed from May 2018 to December 2018. Main Outcomes and Measures: Results of ophthalmic examination, retinal imaging, and molecular genetic analysis by next-generation sequencing. Results: Of 19 patients, 13 (68%) were women, and age ranged from 11 to 70 years. All patients presented with central visual loss, with or without photophobia. Individuals with recessive variants commonly had severe loss of visual acuity by their 20s, whereas the dominant variant was associated with a milder phenotype, with most patients retaining good vision into late adulthood. The recessive cases were associated with a panretinal dystrophy of cone-rod phenotype with early macular involvement, whereas the dominant variants were associated with a cone-rod phenotype that was restricted to the macula with predominantly cone dysfunction. Next-generation sequencing identified 3 novel and 9 previously reported variants in PROM1. Recessive mutations included 6 truncating variants (3 nonsense and 3 frameshift), 4 splice site variants, and 1 missense variant. All 6 dominant variants were associated with a c.1117C>T missense variant. The variants were distributed throughout the PROM1 genomic sequence with no specific clustering on protein domains. Conclusions and Relevance: In this case-series study, PROM1 recessive variants were associated with early-onset, severe panretinal degeneration. The similar phenotypes observed in patients with homozygous missense variants and splice site variants compared with similarly aged patients with truncating variants suggests that all recessive variants have a null (or loss of function close to null) outcome on PROM1 function. In contrast, the dominant missense cases were associated with a milder, cone-driven phenotype, suggesting that the dominant disease is preferentially associated with cones. This has implications for the development of treatments for this severely blinding disease, and adeno-associated viral vector-based gene therapy and optogenetics could become successful treatment options.


Assuntos
Antígeno AC133/genética , Mutação/genética , Degeneração Retiniana/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Distrofias de Cones e Bastonetes/genética , DNA Recombinante/genética , Feminino , Genes Dominantes/genética , Genes Recessivos/genética , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Isoformas de Proteínas/genética , Estudos Retrospectivos , Transtornos da Visão/genética , Adulto Jovem
18.
PLoS One ; 14(4): e0215605, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31002724

RESUMO

Recombinant herpesvirus vaccine vectors offer distinct advantages in next-generation vaccine development, primarily due to the ability to establish persistent infections to provide sustainable antigen responses in the host. Recombinant bovine herpesvirus-4 (BoHV-4) has been previously shown to elicit protective immunity in model laboratory animal species against a variety of pathogens. For the first time, we describe the induction of antigen-specific immune responses to two delivered antigens in the host species after intranasal nebulization of recombinant BoHV-4 expressing the chimeric peptide containing the bovine viral diarrhea virus (BVDV) glycoprotein E2 and the bovine herpesvirus 1 (BoHV-1) glycoprotein D (BoHV-4-A-CMV-IgK-gE2gD-TM). In this study, four cattle were immunized via intranasal nebulization with the recombinant BoHV-4 construct. Two of the cattle were previously infected with wild-type BoHV-4, and both developed detectable serologic responses to BVDV and BoHV-1. All four immunized cattle developed detectable viral neutralizing antibody responses to BVDV, and one steer developed a transient viral neutralizing response to BoHV-1. Approximately one year after immunization, immunosuppressive doses of the glucocorticoid dexamethasone were administered intravenously to all four cattle. Within two weeks of immunosuppression, all animals developed viral neutralizing antibody responses to BoHV-1, and all animals maintained BVDV viral neutralizing capacity. Overall, nebulization of BoHV-4-A-CMV-IgK-gE2gD-TM persistently infects cattle, is capable of eliciting antigen-specific immunity following immunization, including in the presence of pre-existing BoHV-4 immunity, and recrudescence of the virus boosts the immune response to BoHV-4-vectored antigens. These results indicate that BoHV-4 is a viable and attractive vaccine delivery platform for use in cattle.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , DNA Recombinante/imunologia , Herpesvirus Bovino 4/imunologia , Vacinas Sintéticas/imunologia , Administração Intranasal , Animais , Antígenos Virais/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/virologia , DNA Recombinante/genética , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/imunologia , Herpesvirus Bovino 4/genética , Nebulizadores e Vaporizadores , Vacinação/métodos , Vacinação/veterinária , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia
19.
J Vis Exp ; (146)2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-31009004

RESUMO

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.


Assuntos
DNA Recombinante/genética , Engenharia Genética/métodos , Vírus Sincicial Respiratório Humano/genética , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico , Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Genética , Transfecção , Replicação Viral
20.
Int Immunopharmacol ; 70: 467-476, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30861467

RESUMO

CpG oligodeoxynucleotides (CpG-ODN) is an immunoenhancer, which is composed of unmethylated cytosine and guanine. Host Defense Peptides (HDPs) are small molecule polypeptides with various immunological activities that have been shown to induce a stronger innate immune response in piglets with synthetic CpG-ODN. Therefore, combination of CpG-ODN and HDPs was expected to be a novel immunoadjuvant with high efficiency, low toxicity and great potential. However, cost of synthetic HDPs or CpG-ODN is too high to be advantageous for animal farming. In this study, in order to improve the immune function of vaccine and reduce cost, a series of recombinant plasmids (containing HDPs gene (PR-39/pBD-1) and different numbers of CpG motifs) were constructed. In vitro, porcine lymphocytes were stimulated by recombinant plasmids to verify the immunostimulatory function of recombinant plasmids. In vivo, recombinant plasmids were used to immunize piglets with Enterotoxigenic Escherichia coli (ETEC) vaccine to analyze effects of recombinant plasmids on the mucosal immune responses. In addition, dosage screening and capability of maternal antibody responses were also investigated. Our results showed that recombinant plasmids had strong adjuvant effects especially the plasmid pVAX49-PR-39 and pVAX49-pBD-1. Moreover, there was no diarrhea in piglets using pVAX49-PR-39 or pVAX49-pBD-1 as adjuvants. These findings suggested that recombinant plasmids (containing PR-39/pBD-1 and CpG) as adjuvants of vaccines could enhance immune stimulation better than HDPs or CpG alone. It has a good protective effect on maintaining health of newborn piglets. Among them, both plasmids pVAX49-PR-39 and pVAX49-pBD-1 could be used as effective vaccine adjuvants for piglets.


Assuntos
Vacinas Bacterianas/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/fisiologia , Intestinos/imunologia , Suínos/imunologia , Imunidade Adaptativa , Adjuvantes Imunológicos/genética , Agricultura , Animais , Animais Recém-Nascidos , Peptídeos Catiônicos Antimicrobianos/genética , Vacinas Bacterianas/genética , Ilhas de CpG/genética , DNA Recombinante/genética , Feminino , Imunidade Humoral , Imunidade Inata , Imunidade Materno-Adquirida , Imunização , Plasmídeos/genética , Gravidez
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