جستجوی مقالات مرتبط با کلیدواژه « Molecular farming » در نشریات گروه « بیوتکنولوژی و ژنتیک گیاهی »

In the last three decades, molecular farming has emerged as an alternative and cost-effective strategy for the successful production of hundreds of large-scale pharmaceutical recombinant proteins using plants. Because tumor cells need angiogenesis to grow and invade, therefore, targeting the most important angiogenic factor (vascular endothelial growth factor, VEGF) using the plant-based recombinant anti-VEGF can be an effective approach to inhibit angiogenesis and cancer treatment. The low expression level of recombinant protein is one of the major obstacles for the commercialization of molecular pharming. Therefore, numerous techniques have been developed to enhance protein expression, including codon optimization of protein sequences, appropriate subcellular targeting; the use of strong promoters, and the testing of different plant-based expression systems. In this paper, we investigate the effect of apoplast and endoplasmic reticulum (ER) targeting on Anti-VEGF recombinant nanobody accumulation in cucurbit plant. for this purpose, different molecular analyzes (RT-PCR, Dotblot, SDS-PAGE, Westernblot and ELISA) were performed and the results showed that ER targeting increased protein expression about 6-fold compared to its expression in the apoplast. Therefore, the ER is a more suitable site for expression of the target recombinant protein and other recombinant proteins than for apoplast.

Tissue plasminogen activator is one of the most important drugs in the treatment of heart disease. This drug is produced in the expression system as a recombinant protein that has high production costs. Transient expression system is very suitable for protein expression because of its high expression, high speed, low cost and no spatial effect. Post-transcriptional silencing has been shown to affect expression levels. Therefore, the aim of this study was to investigate the effect of simultaneous expression of P19 silencing suppressor gene on transient expression of recombinant tissue plasminogen activator (rtPA) at transcriptional and protein levels in Nicotiana benthamiana. 

To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pCAMBIA1304-rtPA with agrobacterium containing pCAMBIA1304-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pCAMBIA1304-rtPA. Leaf samples were prepared on 4, 7, and 10 day post-inoculation with Agrobacterium. Transcription and then protein levels were calculated using the Real Time PCR and ELISA tests. 

The results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 µg.g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively.

The results showed that the use of transient expression method could be a suitable method for rtPA protein production.

The demand for recombinant proteins with therapeutic use is dramatically increasing; so that the traditional pharmaceutical industry will not be alone to answer the demand for now and upcoming generations. During the past two decades, plant bioreactors has gained more popularity over other conventional methods for several reasons. Among these reasons are scalability, high production rates, low production costs, ability to perform post‐translational modifications, and biosafety of the bioreactor. So far, many important pharmaceutical proteins have been produced using molecular farming technology. In this paper, it has been tried besides a brief description of molecular farming history, types of plant-based systems and molecular farming challenges, appropriate strategies and methods to solve the challenges in this field were being discussed and reviewed.
 

Despite the very promising advances in the field of molecular farming, we still face two serious challenges which needs to be taken seriously; insufficient accumulation levels of recombinant proteins and lack of efficient purification methods. To achieve the high levels of production, several factors should be taken into consideration, such as choice of a suitable promoter or enhancer elements, codon optimization, appropriate subcellular localization, the use of fusion tags and etc. Chromatography methods are routinely used in the pharmaceutical industry for protein purification. Application of these methods has a lot of restrictionsduo to scalability, cost and column pollution problems for purification of plant-derived pharmaceutical proteins.
 

Various fusion protein strategies have been developed not only to increase the yield of plant-derived recombinant proteins, but also to facilitate purification steps.

Today, demand for production of recombinant proteins in different tissues of transgenic plants is increasing day by day. Meanwhile, hairy roots are under consideration due to their high levels of biomass and high genetic stability as appropriate target tissue. Among pharmaceutical proteins, regulatory glycoproteins, are proposed as one of the most important growth factors with therapeutic and research applications in tissue engineering. Selection of proper host species, plant tissue and desirable gene, are of the most important factors to elevate the levels of recombinant protein production. In order to achieve this goal, in the present study, production of recombinant protein BMP2 in transgenic hairy roots of tobacco and canola were investigated. For optimal transgene expression, TMV (5’-UTR) and KOZAK sequences were inserted upstream the BMP2 gene to increase expression level, furthermore, six-histidine sequence for protein purification and KDEL sequence as ER retention signal protein were inserted downstream of the target gene. The transgene was designed to be under the control of CaMV 35S promoter. One-month old tobacco leaf explants and six-day-old cotyledons of canola were transformed with Agrobacterium rhizogenes carrying the construct. Transformed hairy roots that grew well in the medium were confirmed by gPCR analysis for transgene integration. Moreover, to verify mRNA transcription as well as protein production, RT-PCR, ELISA and western blot analyses were performed. The results of this study demonstrated the efficiency of the current systems for the production of rhBMP2 is tobacco hairy roots suggesting them as an efficient bioreactor.

Plants can be exploited for the production of various recombinant proteins as valuable bioreactors because of many reasons. The use of plant viral vectors for the transient expression offers as a useful strategy for the large-scale production of proteins with pharmaceutical importance, such as antibodies within a very short time period. Today, cancer is the second cause of death in human society. Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. In This study, Anti-VEGF (Nb42) nanobody different fragments were transiently expressed under the control of a viral strong promoter in cucurbit plant using ZYMV-based viral vector. These fragments included natural nanobody sequence (Nb42I) and optimized nanobody sequence (Nb42II) for expression in plant. After inoculation of plants, the presence of transcripts of the Nb42 gene fragments in cucurbit inoculated plants was detected by using RT-PCR. Also, the recombinant protein expression was confirmed by Dot blotting, SDS-PAGE, Western blotting and ELISA. Based on the ELISA results, the expression level of Nb42I and Nb42II nanobody fragments was 2 and 6.8 μg/g of leaf tissue respectively. Taken together, recombinant Nb42 nanobody could be efficiently expressed in cucurbit plant and ZYMV-based expression system would be an appropriate platform for production of recombinant proteins in cucurbit plants.

Molecular farming refers to the production of pharmaceutical proteins and industrial enzymes in plants by genetic engineering. T7RNA Polymerase is one of the most important enzymes which is used in various fields of biotechnology and molecular biology to produce RNA and study the structure and function of RNA in vitro. The in vitro synthesized RNA is employed in hybridization, microarray, anti-sense RNA and RNAi experiments. This enzyme has also applications in expression systems depending on the T7 promoter and terminator. T7RNA polymerase is a bacteriophage enzyme which acts mainly in the cytoplasm. This study aimed to clone and transform eukaryotic cells with the T7RNA Polymerase gene for production of T7 polymerase in tobacco plants. Specific primers with proper restriction enzyme sites were designed corresponding to the ends of the T7RNA polymerase gene and the gene was isolated and cloned into the plant expression vector, pCAMBIA1304. Successful of gene insertion was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The recombinant construct was transformed into Agrobacterium tumefaciens LBA4404 strain for tobacco transformation. The transgenic tobacco plants were regenerated on selective media containing hygromycin and cefotaxime antibiotics. The presence and expression of the T7RNA polymerase gene in the transgenic plants was confirmed by PCR and RT-PCR techniques.

Molecular farming is the use of plants to produce valuable pharmaceutical proteins in the bulky and cost-effective manner. Molecular farming and the production of recombinant proteins (such as insulin، interferon-gamma، tissue plasminogen activator، etc.) in transplastomic plants، has achieved a great success in Iran. Due to the growing demand for recombinant proteins، the selection of a proper expression system is very important. Chloroplast genome engineering، for its many advantages، is one of the suitable methods for the production of recombinant proteins. To achieve high levels of recombinant proteins in chloroplasts، plastid’s specific transcription، translation and post-translational modifications should be considered. Some of the strategies used for increasing of foreign proteins in chloroplast can be mentioned: gene insertion in the suitable site in chloroplast genome، use of strong promoters، appropriate regulatory sequences in the 5'' and 3'' UTRs، codon optimization، and utilization of factors enhancing protein stability such as chaperons and fusion proteins. In this article، besides a brief description about chloroplast and its genome، methods for enhancing recombinant protein production in transplastomic plants were discussed.

Plants are suitable replacements for current biodrugs and recombinant protein expression systems such as transgenic animals or microbial systems. As the plant plastid genome is highly polyploid, the transformation of chloroplast permits the introduction of thousands of copies of foreign gene per plant cell and generates extraordinarily high levels of recombinant protein. Cardiovascular diseases are the second one leading cause of human mortality after cancer. Human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of body such as brain and heart blood vessels. The truncated form of tissue plasminogen activator (K2S) has longer plasma half life, better diffusion into the clot and higher fibrinolytic activity. In this study, in order to introduction of K2S gene in tobacco chloroplast, after construct design, the interest gene was ligated to pKCZ vector and cloned in E. coli. Following the successfully shooting of the K2S-containing vector (pKCZK2S) to leaf explants using the biolistic delivery procedure, the explants were selected on selection medium containing 500 mgL-1 spectinomycine antibiotic. After regeneration of grown shoots on selective medium, in order to achieve homoplasmy, fourth rounds of selection and regeneration in presence of spectinomycine antibiotic were performed. The presence, site-specific integration, expression of the K2S gene and homoplasmy in transplastomic plants were confirmed by PCR, RT-PCR and Southern blotting methods.

In the recent decades the progress in plant biotechnology, especially in the field of molecular farming, has made it possible to produce a wide range of recombinant protein using plants as a novel production system and bioreactor. human gamma interferon is one of the valuable pharmaceutical proteins which has found wide medical applications in diagnosis and therapeutic. Cloning of human gamma interferon gene associated with spectinomycinstreptomycin antibiotic resistance gene using chloroplast pKCZ vector has been performed at agricultural biotechnology laboratory of Tarbiat Modares University and has been transferred into the tabacco chloroplast genome with biolistic method. The purpose of this study is to determine gamma interferon gene expression stability in T1 tobacco transplastomic plants. plants, which are grown on selective media, were analysed at different levels of (DNA, RNA and protein) to confirm integration and expression of the gene into the tobacco chloroplast. Analysis of plants was performed at DNA level by using of PCR and specific primers for human gamma interferon gene, results confirmed integration of this gene into the chloroplast genome. In addition, transcription of target gene was confirmed by RT-PCR. In order to study expression of target gene at protein level SDS-PAGE, Dot blotting and Western-blot were also done and the obtained results show target gene expression at protein level. Finally, ELISA test was done to determine quantity of gamma interferon gene expression. Results indicated that the highest gamma interferon level in transplastomic plant is up to 0.2% of total soluble protein.

Interferon gamma (IFN-g) is one of the most important proteins used in diagnostic and therapeutic especially in virus disease therapy and regulation of immune system actions. There are several ways for production of interferon gamma protein as an active ingredient of many medicines and one of these ways is using plants and molecular farming. In latest years chloroplast transformation due to having many advantages such as ability to accumulate large amounts of foreign protein in chloroplast, being free of human pathogens and expression of several genes simultaneously has been considered. The purpose of this research was to check feasibility of interferon gamma production using chloroplast transformation in a dicistronic operon. Interferon gamma gene was cloned in pKCZ vector. Then gold particles coated with this vector were bombarded on tobacco leaves by gene gun. The pieces of leaves were transferred to shoot regeneration medium containing spectinomycin. Thereafter the regenerated shoots were transferred to MS medium for rooting. Presence of interferon gamma gene and it’s expression in regenerated plants was confirmed by PCR and RT-PCR. Additionally gene expression in protein level was confirmed by SDS-PAGE and dot blot. Finally four regeneration rounds were performed to obtain homoplasmy.

The production of pharmaceutical compounds having role in treating diseases like AIDS is necessary. In this regard, griffithsin (GRFT) (an algae lectin) can inhibit HIV entry to the cell at picomolar concentration. To production of recombinant GRFT protein, alfalfa, soybean and lettuce were used as hosts for transient expression. Effect of KDEL, signal peptide of carrot extensin and SP subunit of ZERA signal peptide that target protein to endoplasmic reticulum, apoplastic space and space around the cell wall, respectively was studied. 72 hours after agroinfiltration, qRT- PCR analysis showed high potential of lettuce to express of GRFT at transcript level while alfalfa had lowest transcription level. ELISA result using polyclonal anti-mouse anti-GRFT antibody confirmed that targeting of GRFT to apoplast space of soybean led to highest amount of GRFT. Lettuce despite of high level of gene expression, showed low level of GRFT accumulation which may be due to posttranscriptional and post translational modifications. Our results suggest using of soybean leaf tissue for fast and reliable production of pharmaceutical using transient expression.

the use of plants as a source of medicine is traced back to long time ago. Modern biotechnology proviedes the possibility of production of valuable protein such as pharmaceutical protein in plant. In this study human Interferon gamma-oleosine genes under the control of Napin promoter were transferred to Carthamus tinctorius L. (safflower) by Agrobacterium tumefaciens strain LBA4404. Cotyledonal explants from safflower plant (Carthamus tinctorius L.) Padideh cultivar were used for transformation. The transformed plants were screened on MS medium containing 0.09 mg L-1 NAA, and 1 mg L-1 TDZ containing 40 mg L-1 kanamycin. Presence of transgenes was confirmed using polymerase chain reaction (PCR). Characterization of the transgenic plants is going on.