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The use of different expression plasmids to increase pseudovirus titres using the glycoprotein for rabies virus RV277 - Dissertation Example

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Abstract: Rabies virus is a non segmented RNA containing negative strand and belongs to the Lyssavirus genus and comes from the Rhabdoviridae family. Rabies virus has an envelope protein called G-protein that protects and is responsible for the binding of the virus to the target cells…
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The use of different expression plasmids to increase pseudovirus titres using the glycoprotein for rabies virus RV277
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The use of different expression plasmids to increase pseudovirus titres using the glycoprotein for rabies virus RV277

Download file to see previous pages... The expression level of the gene can be increased by the use of different plasmids. So in order to increase e the expression level of the Rabies virus, the RV277 glycoprotein gene was inserted into the pCAGGS plasmid using the molecular biology techniques such as plasmid DNA isolation, increasing the concentration of the insert DNA using the PCR, restriction digestion of the insert DNA and the plasmid pCAGGS vector, ligation of the insert and the vector using t4 DNA ligase and bacterial transformation of the ligated DNA in E.coli through the heat shock method. The transformed Bacterial cells were found to have positive results indicating that the transformation has worked. The future work of this project includes identification of the gene and further analysis of the effect of transformation of the RV 277 glycoprotein gene. The use of different expression plasmids to increase pseudovirus titres using the glycoprotein for rabies virus RV277 Introduction: Rabies virus is a prototypic and neurotypic member of the Rhabdoviridae family. It is present within the Lyssavirus genus. This virus consists of a 12 Kilo base pair RNA strand which is a negative strand. There are coding for five proteins which are very essential for viral replication and particle formation. This viral RNA is enclosed inside the viral nucleoprotein and has phosphoprotein enclosed in it. The RNA genome contains 2-3% RNA, 67 – 74% proteins, 3% carbohydrates and 20 – 26 % lipids. (Shors 2011). The glycoproteins of the rabies virus are important immunologically as they are capable of reacting very well with the viral particles. It induces and reacts with the viral neutral particles. The surface glycoproteins are responsible for the attachment of the virus to the receptors of the host. They are also responsible for the membrane fusion of the host cell with the vector by the pH mediated fusion. (Shors 2011). The glycopotreins or the G-proteins present in the rabies virus has the ability to accept foreign genes without affecting the rabies viral genome and its functions. Vector based viruses are widely used for delivering the therapeutic genes into the primary cells. HIV viruses are independent of cell division and they are different from the other viruses. The HIV -1 DNA is generally maintained as an extra chromosomal viral DNA in the CD4+ cells and other cells present in the human body. (Reiser et al, 1996). Most of the DNA is reversible transcribed and this transcription is partial. Retro based vectors have the capacity to infect the non dividing cells. The non dividing cells include stem cells, post-mitotic neurons and hematopoietic progenitor cells. If the HIV -1 type is used as a vector system by deleting the viral envelope, then the possibility to use them fruitfully will increase. (Reiser et al. 1996). By inserting the rabies viral envelope gene G- type protein called glycoprotein into the HIV -1 genome , by deleting its own viral envelope gene, the applicability of the vector in primary , non dividing human cells can be determined. (Reiser et al. 1996). Many vectors were developed using the pseudotype vector system. Some scientists concentrated on the two –plasmid system and some on encoding the placental alkaline phosphatase into the nef coding region of the pseudo vectors. The titer value was close to 2 x 10^ 5 / ml. (Reiser et al. 1996). Some scientists worked on the pseudo typing of the VSV- G and HIV-1 vector system. The vectors designed were even capable of transducing the monocyte- ...Download file to see next pagesRead More
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