Controlling CVB3 Particles Using The Genetic Code Expansion

The genetic code expansion is an orthogonal suppressor aminoacyl-t RNA synthetase/t RNA?aa RS/t RNA?identifies the blank code?stop codon or frameshift mutation?introduced in the genome and encodes unnatural amino acid at the corresponding locus.Unnatural amino acids bearing various functional groups are inserted into the protein to obtain a specific site-modified protein.This technology has already showed a variety of application potential in a number of research areas.Coxsackie B3 virus?coxsackievirus B3,CVB3?belongs to the family Picornaviridae.CVB3 is widespread in the human population and causes aseptic Meningitis,pancreatitis and type I diabetes.Although CVB3 has a certain clinical impact,but there are no vaccine or drugs can use into clinical.For the control dilemma of CVB3 and combined with the expansion of the genetic code,this topic envisages to set up a genetic barrier for CVB3 translation and packaging.Amber codon UAG is introduced in the CVB3 genome.In the natural environment,CVB3 translation will be stop due to the introduction of amber mutations and premature termination.While in artificial environment,aa RS/t RNA can correctly identify the amber mutations introducted and encode unnatural amino acid at the corresponding sites,the translation of CVB3 can continue and package a complete virus.The CVB3 mutants constructed in this study are controllable which encapsulated in artificial environment is immunogenic,but can not be amplified in the natural environment.It provides a new train of thought to develop CVB3 vaccine.This research selected Pyl RS/t RNA_CUA which is derived from Methanosarcina mazei.Pyl RS does not exist in eukaryotic cells.The selected Boc-lysine is artificially synthesized and does not exist in the natural environment.So it further enhance the controllability and safety of CVB3.The main contents and results are as follows:1.Based on the survey,we chose Mm Boc RS and U6-Pylt RNA_CUA from Methanosarcina mazei.Literature review and Gene Bank alignment to determine the final fragment sequence before gene synthesis was performed.The Mm Boc RS gene fragment was ligated into vector pc DNA3.1,and then four copies of U6-Pylt RNA_CUA fragmentswas inserted.They were named Mm Boc RS-pc DNA3.1,Mm Boc RS-t RNA,Mm Boc RS-2t RNA,Mm Boc RS-3t RNA and Mm Boc RS-4t RNA.2.The above constructed plasmid was transiently transfected into HEK 293 T cells.Western Blot showed that Mm Boc RS could express normally in the cells and Pylt RNA_CUA expressing in the cells was verifiably real-time quantitative PCR.The expression of Pylt RNA_CUA was increased with the augment of copy numbers.The results showed that the plasmids of the extended codon could be expressed normally in the cells.3.The EGFP mutant plasmid p EGFP-N1-UAG was constructed by inducing mutant at the 39 position of p EGFP-N1.p EGFP-N1-UAG and the genetic codon expansion system were co-transfected into the cells.The fluorescence microscope and western blot showed that the genetic code expansion plasmid has the function normally in HEK 293 T cells and could control protein expression by unnatural amino acid Boc-lysine.Furthermore,the expression rate of unnatural amino acid was further analyzed by flow cytometry.The green fluorescent protein of p EGFP-N1-UAG was not increased as expected with the increase of Pylt RNA_CUA copy number.Compared with wild-type EGFP,the insertion efficiency of Boc-lysine is 65.20%.4.Amber mutants were introduced at different positions of CVB3 genome.A total of 16 CVB3 mutants were constructed successfully.CVB3 amber mutant plasmid was co-transfected with Mm Boc RS-4t RNA plasmid and the transfected product was subjected to repeat freezing-thawing.The virus was harvested and the next generation of cells was infected.CVB3 expressed of the two generations was detected by real-time quantitative PCR analysis.The results showed that the addition or absence of Boc-lysine could make the expression of CVB3 amber mutant significantly difference,in the same word the virus amplification was controlled by unnatural amino acid.From viral RNA extraction,reverse transcription,PCR to and sequence the amplification products,it found that some of the CVB3 amber mutants reverted mutated.5.In order to ensure the temporal and spatial consistency of each test and to avoid the factors such as cell status,cell density and experience of the experimenter,the lentiviral packaging technique was used to construct stable cells Boc RS-t RNA_CUA that could express the genetic codon extension system.Western blot and real-time quantitative PCR were performed on stable cells,and further transfection of p EGFP-N1-UAG was performed.The results showed that Mm Boc RS and Pylt RNA_CUA could be expressed stably in Boc RS-t RNA_CUA cell,and green fluorescence showed that the stable cell line could have the function of genetic codon extension.Considering the poor efficiency of expression,it need to be further optimized.In conclusion,the CVB3 amber mutant was constructed and a genetic barrier was set up for CVB3 through genetic code expansion and unnatural amino acid Boc-lysine.The artificial environment and natural environment were isolated.The CVB3 amber mutant could translation artificial conditions,in natural conditions could not self-replication but did not lose the original immunogenicity of the virus.It can provide a new train of thought for development the vaccine of CVB3 and other small RNA virus.The stable cell line Boc RS-t RNA_CUA couldfacilitated the subsequent research of genetic codon expansion.