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Adenosine deaminase deficient severe combined immune deficiency is an autosomal recessive metabolic disorder caused due to the absence of purine salvage enzyme adenosine deaminase. In purine salvage pathway ADA plays an important role by degrading adenosine and deoxyadenisine into inosine and deoxyinosine respectively. So the deficiency of this enzyme results in the accumulation of toxic metabolites, which adversely affects the proper functioning and development of lymphocytes. This condition will lead to life threatening susceptibility to various infectious diseases and in most cases death will occur within few months if left untreated,(Belinda et al.,2005).
There are currently three main therapeutic treatments are available for this metabolic disorder, which are bone marrow transplantation from a histocompatiable sibling, gene therapy and enzyme replacement therapy Among these three therapeutic treatments, enzyme replacement therapy using ADA from a bovine source coupled with PEG, is delivered to patients through intramuscular injection is found to be more effective due to its rapid metabolic detoxification and long team immune recovery. Although this method has got approval from FDA, several side effects including haemolytic anaemia, autoimmune haemolytic anaemia, thrombocytopenia, erythema and so on has been reported it patients. (Ochs et al., 1999)
However the exact problem behind this side effects remains unclear, the treatment replacing bovine ADA with human ADA will be more effective for curing ADA deficiency. This project particularly aims to express human ADA in transgenic plants.
Transgenic plants represents an excellent expression system for the production of therapeutically important recombinant proteins,( Twyman et al.,2003)
compared to conventional expression systems, the main advantages offered by plant expression systems are the ability to perform post translational modification including proper mechanism of protein folding and disulphide bridging (Daniell et al.,2001), low rate of contamination due to the absence of human pathogens, oncogenic DNA sequences and endotoxins (Twyman et al.,2003), large scale cost effective production, ease of scalability, elimination of expensive down stream processing when expressed in edible plant tissues. (Shih and Doran., 2009).
In addition to this, protein production can be targeted to a desirable part or organelle of transgenic plant by using specific promotor for enhancing expression level (Larrick and Thomas., 2001)
Plant cell culture also act as an efficient and alternative production platform for expressing recombinant protein. In this approach plant cell isolated from suitable host species is cultivated under sterile invitro conditions to express heterologous proteins(Shih and Doran.,2009).
Most of the studies for expressing recombinant proteins are now being conducted using plant tissue culture due to its manifold advantages. De differentiated cell suspension and callus cultures are more appropriate for this sort of studies (Doran., 2000). Issues regarding segregation of gene and transgene instability can get rid of by using plant culture system. Ease of rapid manipulation And maintenance under sterile environment, adequate control over protein expression level and product quality, excellent and consistent production with in short duration of time, simple and inexpensive purification are other merits of this system(Shih and Doran.,2009). This is well exemplified by successful production of recombinant antibodies and fusion proteins in BY-2 tobacco and rice cell suspension cultures (Fischer et al., 1999).
Many therapeutically important and commercially valuable recombinant proteins were successfully produced in plants. Some of which are Sig A anti-S mutans antibody, lymphoma idiotypes, Hep B surface antigen, rabies vaccine , growth hormone, collagen, erythropoietin in transgenic tobacco; lactoferrin, cholera toxin B and E-coli toxin in transgenic potato; IgG antiherpes simplex virus in transgenic soyabean; various anti bodies , anti carcino embryonic antigen and lipase in transgenic cereal plants.(Larrick and Thomas.,2001).
There are variety of host plants are available for transformation they are mainly leafy crops, cereals, fruits and vegetables. The main leafy crops used are tobacco and alfalfa. Compared to all other plant specious tobacco has a long team history of successful protein production. The main advantages of transgenic tobacco are maximum yield, well developed technology for transformation and gene expression, increased level of total soluble protein, low risk of food chain contamination(Tremblay et al.,2010).
But the high level toxic alkaloid content present in tobacco may sometimes interfere with downstream processing. However this problem can overcome by using tobacco cultivar '81V9' which has low nicotine and alkaloid content. (Rainer et al., 2004). The first plant derived therapeutic protein, human serum albumin was produced in transgenic tobacco (European Molecular Biology Organization)
Tobacco offers different expression strategies for recombinant protein production. They are stable nuclear transformation, chloroplast transformation and transient expression.Transformation of nuclear genome can be done by agrobacterium infection or biolystic delivery.this method ensures continuous production of heterologous proteins with minimum cost proteins undergo typical post translational machinery get accumulated in various organelle. But compare to chloroplast, nuclear transformation has low level protein accumulation (Tremblay et al., 2009)
Increase the level of protein production can be achieved by using chloroplast expression system due to its large size and number.(Tremblay et al., 2009). This is well demonstrated by the production of CTB insulin fusion in chloroplast, which showed 160 fold increase in protein accumulation than compared to the same expressed in nuclear genome .(Rahlman et al., 2007). One significant advantage of chloroplast gene expression is the minimum risk of containment through pollen owing to the property of maternal inheritance the main commercially valuable recombinant proteins produced in transgenic tobacco chloroplast includes human insulin like growth factor, canine parvo virus vp2 Antigen, Human Serum Albumin, human interferon α and Anti microbial peptide(Daniell et al., 2004).
Transient gene expression facilitates rapid production of recombinant protein with in short duration of time. Here expression of foreign protein is achieved by using viral vectors. In this approach cDNA clones are generated from plant RNA viruses by reverse transcription and RNA transcripts are produced. These RNA viruses infects plant cells and integrates its genetic material into the cell cytoplasm, not to plant genome.replication of these infected viruses with in the plant cell results in transient expression and accumulation of foreign protein with in plant cell(Shih and Doran.,2009).
There exists an alternative strategy for transient gene expression using viral vectors. In this method plant leaves are filtrated in vaccum with Agrobacterioum tumefaciens containing the inserted DNA sequences.This method enables bacteria to enter plant tissue and there by facilitates DNA transfer(Kapila et al.,1997;vaquero et al1999).
There are several strategies for ensuring optimum production of recombinant protein with in plants. Protein production can be optimized at each steps of gene integration, transcription, translation and protein accumulation(priti et al 2010).selection of suitable promoter also plays an important role in enhancing protein production due it's impact on transcription. For dicot plants strong constitutive CaMV35s promoter is used , but for cereal plants it is not effective and so maize ubiquitin -1 promoter is used. In maize plant high level transcription can achieved by the introduction of an intron and this phenomena is known as intron mediated enhancement. Translation is also optimized by removing mRNA instability sequences from trans gene construct, by ensuring the translation site is similar to kozak consensus for plants(Reynald et al.,2010.
The main tools for expression of heterologous protein are gene or complementeary DNA encoding the protein of interest, suitable vector,suitable expression cassette for transcription and translation of transgene(Priti et al.,2010).Several strategies for expression of recombinant protein,design of gene construct,suitable vectors of choiceand different transformation methods can further be understand by focusing on the production of heterologous protein expressed on transgenic tobacco and chlorolplasts.
A clone of HvBADH1 gene from Hulles Barley has been expressed in transgenic tobacco. Here a cDNA which codes for Betaine aldehyde dehydrogenasc can be produced by reverse transcription and cloned by RT-PCR and RACE. After cloning , an open reading frame of BADH gene is introduced in to the T-DNA region of binary vector pCAMBIA1301 between CaMV35s promoter and Nos poly A. It is then transformed into Nicotiana tobacum via agro bactrerioum tumefaciens (wei et al., 2008).
Another recombinant protein expressed in tobacco cells is HIV Nef variant. It used CaMV35S promoter, nopaline synthase terminator, pBI121 binary plant expression vector, p25 and p27 construct. A specific enhancer and tobacco mosaic virus 5' leader sequence. The construct is introduced in to argo bacterium tumefaciens strain LBA4404. Then tobacco cells are transformed by leaf disk transformation.
For expressing human cytomegalo virus glyco protein in tobacco plants, a binary vector pRD400 is produced which targets protein production to the seeds of tobacco plants. The vector contains gene NPT II for kanamycin resistance, cDNA for gB, NOS-T termination signal ,right and left border sequences of TDNA for enabling transfer into agro bacterium tumefaciens here the resulting plasmid is transferred into agro bacterium strain LBA4404 then tobacco leaf disks are transformed by using this binary vectors and transformed callus cultures are selected on medium containing kanamycin resistance (Tackaberry et al., 1999).
One important recombinant protein produced in tobacco is human actylcholine esterase . The enzyme is expressed and produced in hairy root organ cultures of transgenic nicotaina benthamina plants by agro bacterium mediated transformation. Hairy root cultures is used as an expression system due to genetic and biochemical stability. In this approach agro bacterium rhizogenes is used for transformation of explants. Here cDNA of acetylcholine esterase is produced by denovo synthesis.code transformation of nicotaina benthamina explant with co-transfermed cellline is carried out by using agro bacterium rhizogenes.
However one disadvantage of plant expression system is its inability to carry out proper glycoslation of protein. but this problem can overcome by using invitro modification of recombinant protein by using purified β 1,4 galactosyl transferase and sialyl transferase enzyme(reynald et al., 2010).
Materials and methods:
Selection of host plant:
tobacco is the most common plant of choice for expressing recombinant protein owing to its easy manipulation and transformation, increase in bio-mass production under both invitro and green house conditions and so on. In exception to this tobacco is the most suitable platform for chloroplast transformation. nicotiana tabaccum or nictiana benthamina can be selected host plant of choice. inctiana benthamina is serve as an excellent plant of choice for transient expression of foreign proteins.(Joensuu et al.,--)
There are different plant transformation methods exist Of which agro bacterium based binary expression vector mediated transformation is most common. In this approach the desired DNA is first introduced in to agro bacterium tumefaciens and the bacteria then transforms plant tissue. The introduction of DNA using electroporation into protoplast of plant, PEG mediated uptake of DNA, calcium phosphate mediated co-precipitation of particle bombardment by using gold particle, transient expression using viral vectors chloroplast transformation are the other transformation processes(Franken et al.,1997).
Production of plasmid construct :
Production of plasmid construct needs complementary DNA which codes for adenosine deaminase cDNA can be cloned by polymerase chain reaction to generate gene construct. Suitable vector is needed to produce agro bacterium based primary expression vector. pB19, is consider to be non enveloped icosahaedral human virus with 9135 base pair size, is most suitable for the production of binary vector (Zhi et al.,2003).
The desired DNA clone is introduced in to pB19 vector, nopalinesynth ase promoter and strong constitutive promoter CaMV35s promoter can be used for enhancing expression (Yang et al.,2009)
Left and right border sequences of T-DNA is also included in the construct to ensure gene delivery in to agro bacterium tumefaciens in addition to this npt II (Neomycin phosphotransferase ) gene is used for selection of transformants in MS medium containing kanamycin. Npt2 confers resistance to kanamycin in plants. Nopaline synthase terminator sequence can also used as terminator gene(Joensuu et al., 2010).The gene construct is sequenced to eliminate errors during PCR(Yang et al2009).The gene construct is cloned into pB19 and a binary expression vector is produced.This binary expression vector is then transferred into agrobacterium tumefaciens strainLBA4404 by electroporation(joensuu et al).
Transformation of tobacco
Agrobacterium tumefaciens is transformed into tobaccoby leaf disc method.The transformed plant s is selected from MS medium containing kanamycin.The regeneration of callus tissue occur with in 3-4 weeks. The regenerated shoot is then transferred into root inducing media. From there plant is transferred in to green house conditions
Isolation and purification of ADA
The expressed ADA gene can be isolated from plant tissue using appropriate protocol and then purification is carried out using electrophoresis.
Characterization of human ADA
The extracted ADA gene can be amplified and characterized using polymerase chain reaction and restriction enzyme mapping.
Production of human ADA will be more effective therapeutic treatment for ADA deficiency the human ADA gene can produce in transgenic tobacco. There are different approaches for expressing human ADA in transgenic plants. Compare to all other expression systems tobacco plant is found to be more suitable for recombinant protein production.