EXPRESSION OF THE SURFACE ANTIGEN BRUCELLA ABORTUS OMR16 IN NICOTIANA BENTHAMIANA PLANT

Brucellosis is one of the most contagious and infectious diseases with high incidence rates of cattle and humans in Kazakhstan. Using modern biotechnology techniques to develop vaccines that are reliable and affordable for farmers is an alternative solution to the problem. Plant viruses are often used as a vector for obtaining the expression of antigens of the pathogen. The grape virus A (BAB) is widely used among plant viruses. Brucella membrane proteins are the main objects of this research for futher development of vaccines or diagnostic texts against brucellosis, Membrane proteins (OMPs) are cell specific surface antigens that are immunogenic. OMPs are ideal candidates for the production of recombinant brucellosis vaccines. The object of the study was the outer membrane protein (Omp16), which plays an important role in the suppression of TNF-α production in macrophages. In this study, molecular cloning and analysis of the expression of the Omp16 gene, which was used to express the recombinant protein in plants, was carried out. We selected brucella from the vaccine strain of Brucella abortus 19, and the plant Nicotiana benthamiana, as the subjects for our research, since they widely used for the production of recombinant proteins, and they both appropriate for molecular genetic research. A viral vector was constructed to express the brucellosis antigen Omp16 in Nicotiana benthamiana plants. Nineteen explants were used for the regeneration of transgenic plants. As a result of this studies, the introduced gene of Omp16 was under the subgenomic promoter control of the ORF4 and was successfully expressed while maintaining the efficiency of expression in transgenic plants. The efficiency of viral vectors was evaluated at the level of transcription during expression of the protein Omp16 with viral proteins. The entire leaf blade was infiltrated; the density of Agrobacteria was 0.7. We were able to obtained transgenic plants Nicotiana benthamiana carrying the gene of capsid protein BAB, and the expression of the membrane antigen Omp16 in the viral vector was achieved by replacing the ORF4 with the Omp16 gene. The development of transgenic plants was carried out using agrobacterial transformation.

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The ORF1 Products of Tombusviruses Play a Crucial Role in Lethal Necrosis of Virus-Infected Plants

Journal of Virology ◽

10.1128/jvi.74.23.10873-10881.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 10873-10881 ◽

Cited By ~ 35

Author(s):

József Burgyán ◽

Csaba Hornyik ◽

György Szittya ◽

Dániel Silhavy ◽

György Bisztray

Keyword(s):

Transgenic Plants ◽

Viral Replication ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Crucial Role ◽

Viral Vector ◽

Potato Virus X ◽

Wild Type ◽

Systemic Necrosis

ABSTRACT Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided intrans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.

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660 Production of Therapeutic Proteins in Transgenic Plants and Viral Vectors

HortScience ◽

10.21273/hortsci.35.3.512b ◽

2000 ◽

Vol 35 (3) ◽

pp. 512B-512

Author(s):

John Hammond

Keyword(s):

Transgenic Plants ◽

Cell Lines ◽

Viral Vectors ◽

Human Monoclonal Antibody ◽

Therapeutic Proteins ◽

Plant Viruses ◽

Eukaryotic Expression ◽

Animal Pathogens ◽

Hiv 1 ◽

Glycosylated Proteins

Transgenic plants and plant viruses have potential advantages over other production systems for therapeutic proteins. 1) Plants are not susceptible to human and animal pathogens, such as viruses that can contaminate mammalian and avian cell lines used for production of many vaccines. Recent experiences of “mad cow” disease and theories of the possible origin of HIV from monkey cell lines have highlighted the need for increased product safety. 2) There are established protocols for preparing naturally occurring pharmaceuticals from plants. 3) Unlike bacteria, plants recognize the same glycosylation signals as other eukaryotic expression systems such as mammalian, insect, or yeast cell cultures and can thus produce glycosylated proteins. Although there are differences between plants and other eukaryotes in the types of sugar residues added to glycosylated proteins, it has been demonstrated several times that plant-produced proteins have similar stability and bioequivalence of function and that antigenicity is similar. 4) Plants can produce high yields; a single transgenic plant could yield as much human glucocerebrosidase as 500 placentae. We expressed an epitope from HIV-1 on the surface of bean yellow mosaic potyvirus (BYMV) coat protein (CP); protein produced in transgenic plants is recognized by a human monoclonal antibody that neutralizes most HIV-1 isolates. Epitope-modified BYMV-CP can be recovered from transgenic plants by incorporation into BYMV virions following infection of the transgenic plants. Modified virions display the HIV-1 epitope in a semi-regular array that should stimulate the immune system to a greater degree than free subunits. HIV epitope-bearing BYMV has been used to immunize mice to assess the immune response.

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The estimate of the rubella elimination status in the Russian Federation in 2019

Russian Journal of Infection and Immunity ◽

10.15789/2220-7619-atr-1663 ◽

2021 ◽

Author(s):

T. Chekhlyaeva ◽

O. Czvirkun ◽

N. Turaeva ◽

D. Erokhov ◽

L. Barkinkhoeva ◽

...

Keyword(s):

Russian Federation ◽

Molecular Genetic ◽

Genetic Research ◽

Incidence Rates ◽

Genetic Monitoring ◽

Successful Implementation ◽

Reference Laboratory ◽

Regional Office ◽

The Russian Federation ◽

Endemic Transmission

In 2002, the WHO Regional Office for Europe developed a Strategic Program for the Prevention of Measles and Congenital Rubella Infections in the European Region, which was revised in 2004. As a result of the revision, an additional target was set to eliminate endemic rubella in the region by 2010. Rubella is a disease that is well controlled by vaccination, which will determine the theoretical possibility of interrupting its global transmission. Since 2013, the Russian Federation has been implementing the National Rubella Elimination Program. Elimination criteria have been revised as the Program progresses. Currently, the main criterion for rubella elimination is the absence of endemic (local) transmission of the virus for at least 36 months, which should be confirmed by molecular genetic research methods. In addition, in the Russian Federation, an incidence rate of less than 1 case per 1 million population is also used as one of the elimination criteria. The successful implementation of the Program is supported by the fact that since 2013, against the background of a high (over 95%) coverage of preventive vaccinations, there has been a decrease in incidence rates and their stabilization at a level of less than 1 per 1 million population since 2014. Genetic monitoring of rubella virus strains circulating among the population showed the termination of endemic transmission of the virus. During the implementation of the Elimination Program, the prevailing genotypes of the virus circulating in Russia were genotypes 1E and 2B, which have a global distribution. The data obtained from the results of molecular genetic monitoring made it possible to determine that the strains isolated during the period under consideration belong to different clusters, which speaks in favour of their imported character. Considering the above factors: high vaccination coverage, low incidence and lack of endemic transmission of the virus, the WHO Committee on verification of measles and rubella elimination in 2017 awarded the Russian Federation the status of a country that has achieved rubella elimination. The continuation of the phase of elimination of infection is confirmed annually. This article presents the results of a comprehensive assessment of the rubella elimination status in the Russian Federation by specialists from the National Scientific and Methodological Center for Measles and Rubella and WHO EURO Moscow regional reference laboratory for measles and rubella based on epidemiological data and data from molecular genetic studies in 2019.

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VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

Plant Physiology ◽

10.1093/plphys/kiab197 ◽

2021 ◽

Author(s):

Arjun Khakhar ◽

Cecily Wang ◽

Ryan Swanson ◽

Sydney Stokke ◽

Furva Rizvi ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Viral Vectors ◽

Viral Vector ◽

Tobacco Rattle Virus ◽

Plant Size ◽

Great Promise ◽

Attractive Alternative ◽

Gibberellin Biosynthesis ◽

In Planta

Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work we developed a technology called VipariNama (ViN) in which vectors based on the Tobacco Rattle Virus (TRV) are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

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How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

International Journal of Molecular Sciences ◽

10.3390/ijms22147545 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7545

Author(s):

Myriam Sainz-Ramos ◽

Idoia Gallego ◽

Ilia Villate-Beitia ◽

Jon Zarate ◽

Iván Maldonado ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Practice ◽

Gene Delivery ◽

Viral Vectors ◽

Viral Vector ◽

Clinical Success ◽

Genetic Material ◽

Viral Gene ◽

Promising Alternative ◽

Vector Systems

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

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Population Genetic Structure Analysis Reveals Decreased but Moderate Diversity for the Oriental Fire-Bellied Toad Introduced to Beijing after 90 Years of Independent Evolution

Animals ◽

10.3390/ani11051429 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1429

Author(s):

Yang Teng ◽

Jing Yang ◽

Guofen Zhu ◽

Fuli Gao ◽

Yingying Han ◽

...

Keyword(s):

Genetic Structure ◽

Structure Analysis ◽

Phylogenetic Trees ◽

Molecular Genetic ◽

Genetic Research ◽

Haplotype Diversity ◽

Botanical Garden ◽

Source Population ◽

Independent Evolution ◽

Loop Region

Detailed molecular genetic research on amphibian populations has a significant role in understanding the genetic adaptability to local environments. The oriental fire-bellied toads (Bombina orientalis) were artificially introduced to Beijing from Shandong Province in 1927, and since then, this separated population went through an independent evolution. To explore the differentiation of the introduced population with its original population, this study analyzed the genetic structure of the oriental fire-bellied toad, based on the mitochondrial genome control region and six microsatellite sites. The results showed that the haplotype diversity and nucleotide diversity of the mitochondrial D-loop region partial sequences of the Beijing Botanical Garden population and the Baiwangshan population were lower than those of the Shangdong Kunyushan population. Microsatellite marker analysis also showed that the observed heterozygosity and expected heterozygosity of the Beijing populations were lower than those of the Kunyushan population. The phylogenetic trees and network diagrams of haplotypes indicated that the three populations were not genetically separated. However, the structure analysis showed a genetic differentiation and categorized the sampling individuals into Beijing and Shandong genetic clusters, which indicated a tendency for isolated evolution in the Beijing population. Although the Beijing populations showed a decline in genetic diversity, it was still at a moderate level, which could maintain the survival of the population. Thus, there is no need to reintroduce new individuals from the Kunyushan source population.

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Somatic embryo induction and Agrobacterium-mediated transformation of embryonic callus tissue in Phoebe bournei, an endangered woody species in Lauraceae

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha48211946 ◽

2020 ◽

Vol 48 (2) ◽

pp. 572-587

Author(s):

Wenting XU ◽

Miao ZHANG ◽

Chen WANG ◽

Xiongzhen LOU ◽

Xiao HAN ◽

...

Keyword(s):

Genetic Transformation ◽

Germination Rate ◽

Molecular Genetic ◽

Genetic Research ◽

Woody Species ◽

Technical System ◽

Transformation Rate ◽

Original Material ◽

Embryonic Callus ◽

Phoebe Bournei

Phoebe bournei, a plant species endemic to China, is a precious timber tree and widely used in landscaping. This tree contains numerous secondary metabolites, underscoring its potential economic value. However, studies on this species, including molecular genetic research, remain limited. In this study, both a somatic embryogenesis (SE) technical system and Agrobacterium-mediated genetic transformation were successfully employed in P. bournei for the first time. The SE technical system was constructed using immature embryos as original material. The primary embryo and embryonic callus induction rates were 30.66% and 41.67%, respectively. The highest rate of embryonic callus proliferation was 3.84. The maximum maturity coefficient and germination rate were 53.44/g and 39%, respectively. Agrobacterium-mediated genetic transformation was performed using the SE technical system, and the highest transformation rate was 11.24%. The results presented here are the first to demonstrate an efficient approach to achieve numerous P. bournei plantlets, which serves as the basis for artificial cultivation and resource conservation. Furthermore, the genetic transformation platform constructed in this study will facilitate assessment of gene function and molecular regulation.

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Chloroplast Hsp70 Isoform Is Required for Age-Dependent Tissue Preference of Bamboo mosaic virus in Mature Nicotiana benthamiana Leaves

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0012-r ◽

2017 ◽

Vol 30 (8) ◽

pp. 631-645 ◽

Cited By ~ 3

Author(s):

Ying Wen Huang ◽

Chung Chi Hu ◽

Ching Hsiu Tsai ◽

Na Sheng Lin ◽

Yau Heiu Hsu

Keyword(s):

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Transit Peptide ◽

Plant Viruses ◽

Age Dependent ◽

Efficient Replication ◽

Underlying Mechanisms ◽

Lines Of Evidence ◽

Suitable Environment

Plant viruses may exhibit age-dependent tissue preference in their hosts but the underlying mechanisms are not well understood. In this study, we provide several lines of evidence to reveal the determining role of a protein of the Nicotiana benthamiana chloroplast Hsp70 (NbcpHsp70) family, NbcpHsp70-2, involved in the preference of Bamboo mosaic virus (BaMV) to infect older tissues. NbcpHsp70 family proteins were identified in complexes pulled down with BaMV replicase as the bait. Among the isoforms of NbcpHsp70, only the specific silencing of NbcpHsp70-2 resulted in the significant decrease of BaMV RNA in N. benthamiana protopalsts, indicating that NbcpHsp70-2 is involved in the efficient replication of BaMV RNA. We further identified the age-dependent import regulation signal contained in the transit peptide of NbcpHsp70-2. Deletion, overexpression, and substitution experiments revealed that the signal in the transit peptide of NbcpHsp70-2 is crucial for both the import of NbcpHsp70-2 into older chloroplasts and the preference of BaMV for infecting older leaves of N. benthamiana. Together, these data demonstrated that BaMV may exploit a cellular age-dependent transportation mechanism to target a suitable environment for viral replication.

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