Difference between revisions of "Part:BBa K3504009"

Revision as of 17:42, 21 October 2020

Alphavirus replicon NSPs- Equine Encephalitis virus

Part Description

A composite of parts (BBa_K3504000,BBa_K3504001,BBa_K3504002,BBa_K3504003) and CMV promoter which form ,as a unit, the main constituent of alphavirus replicon which as a whole can give the circuit self-replicating ability

Usage

Alphavirus genomes encode four non-basic proteins, nsP1–4, which are translated from the genomic RNA and interact with host factors to form replicative enzyme complexes. These non-structural proteins are translated as a polyprotein that self-cleaves into separate proteins, which assemble into a replicase complex. The replicase then directs replication and amplification of our vaccine inside the Myocytes.

References:

Maruggi, Giulietta, et al. “Engineered Alphavirus Replicon Vaccines Based on Known Attenuated Viral Mutants Show Limited Effects on Immunogenicity.” Virology, vol. 447, no. 1–2, Dec. 2013, pp. 254–264, 10.1016/j.virol.2013.07.021. Accessed 25 Sept. 2020.

https://openarchive.ki.se/xmlui/bitstream/handle/10616/42305/Thesis_Maria_Lisa_Knudsen.pdf;sequence=3

Characterization

Figure 1.in vitro replicon enhancement method: Using transfected Jurkat cells with replicon RNA which was encoded for mCherry and grown in cell culture under the SGP. Then during serial passage as shown in the flow cytometry histograms, the top 20 percent of mCherry were sorted around every 10 days. this has resulted that cells expressing higher levels of repoter gene were enriched. finally the 5th sort cells were seperated from the rest for replicon sequencing.

Figure 2.Mutation identification:Positive mCherry cells was reverse transcribed to cDNA,after that Nsp1 to 4 and the SGP were magnified by seven pairs of primers and amplicons and later on engineered into DNA of the plasmid and changed into E.coli to be amplified. The lower left part schematic illustrates roughly the locations of point mutations in the 5th sort in NSP2 & NSP3.

Improvements

Using information in literature we were able to increase the replicon cloning and functional ability by adding G110G, G763R Mutation to NSP2 and adding K94E, S243G,E255D,V305M Mutation to NSP3

Figure 1. Nsp2 G110G Amino acid mutation.

Figure 2. Nsp2 G763R Amino acid mutation.

Figure 3. Nsp3 K94E Amino acid mutation.

Figure 4. Nsp3 S243G Amino acid mutation.

Figure 5. Nsp3 E255D Amino acid mutation.

Figure 6. Nsp3 V305M Amino acid mutation.

References

Li, Y., Teague, B., Zhang, Y., Su, Z., Porter, E., Dobosh, B., ... & Weiss, R. (2019). In vitro evolution of enhanced RNA replicons for immunotherapy. Scientific reports, 9(1), 1-10. Sequence and Features