Construction Of An In Vitro Transcription And Delivery System For Feline Coronavirus S Protein Fragment MRNA

Feline coronavirus(FCoV)is a single-stranded positive-stranded RNA virus.there are two biotypes of FCoV,namely feline enteric coronavirus(FECV)and feline infectious peritonitis virus(FIPV).virus(FIPV).Among them,FIPV causes the highly lethal feline infectious peritonitis(FIP),which can lead to feline mortality.In addition,FCoV can be divided into two genotypes,type I and type II,which are present in both FECV and FIPV biotypes.In China,the type I FCoV strain has a high prevalence in both FIPV-infected cats and clinically healthy cats.Although the pathogenesis,transmission pattern and comprehensive control of FCoV have been explored both at home and abroad.However,prevention techniques for FCoV remain limited and are generally only symptomatic treatment after the onset of the disease.Moreover,the in vitro culture of FCoV is difficult,resulting in a relative lag in basic research and vaccine research on FCoV,and no safe and effective vaccine is available on the market.mRNA vaccines have been used in the prevention and control of many infectious diseases.mRNA vaccines do not carry dangerous components of viruses,have no risk of infection,have short development cycles,can be developed rapidly in response to virus mutations,can stimulate both humoral and cellular immunity,are immunogenic,do not require adjuvants and can be easily mass produced.Especially with the rapid evolution of SARS-CoV-2 virus and the emergence of new pathogens worldwide,mRNA vaccines are more widely used than ever before.Thus,mRNA vaccines have great potential for use in the prevention of cancer and viral diseases.The coronavirus S protein plays an important role in virus infection and antibody induction,and the selection of FCoV S protein in vaccine development is more favorable for its vaccine development and diagnostic technology development.Our group has expressed the type I FCoV S protein fragment in vitro,and immunized mice with the purified protein and performed ELISA assay to show that the recombinant protein has good immunity effect,which proves that this S protein fragment has the ability to be used as a candidate antigen for vaccine preparation.In order to construct an mRNA vaccine that can efficiently express the FCoV S protein gene,the S protein fragment obtained above was selected for gene modification in this study.Three in vitro transcription models,named IVT-mRNA-n1,IVT-mRNA-n2 and IVT-mRNA-n3,were constructed according to the different sequencing of non-coding regions(UTRs),and their effects on the translation efficiency of mRNA were analyzed.The three constructed mRNA in vitro transcription models were inserted into the in vitro transcription vector p GEM-3Zf(+)to construct the PVAX1-S recombinant plasmid,and transformed into E.coli TOP10 mass culture to extract the recombinant plasmid plasmids.The recombinant plasmid was prepared by Xba1 monoenzymatic cleavage into a linearized DNA template and transcribed in vitro into FCoV S mRNA.In addition,given that the green fluorescent protein EGFP has the feature of easy observation and detection,the same method was used to construct p GEM-EGFP plasmid and transcribed in vitro into EGFP mRNA in this study.the best in vitro transcription system was screened using S protein and EGFP.The results of Western Blot assay showed that both S protein and EGFP showed the highest protein expression on IVT-mRNA-n1,indicating that IVT-mRNA-n1 was screened for the optimal in vitro transcription system.In addition,the results showed that IVT-mRNA-n1 expressed S protein gene in several different cell lines(CRFK,FCWF-4,293 and PK-15),indicating that this system has the potential to be applied in different cell lines.In response to the prevalence of ribonucleases(RNases)in the environment,cationic lipid nanoparticles(LNPs)were synthesized in this study.The nanoparticles can protect the mRNA vaccine from degradation,so that it can reach the ribosome and be translated into antigenic proteins smoothly.In this study,EGFP mRNA was used as a reporter gene to map the conditions of the mRNA-LNPs protection delivery system,and the mapped LNPs protection system was subsequently self-assembled with the constructed S mRNA to form S mRNA-LNPs vaccine,and the particle diameter,potential size,and cytotoxicity were measured.The results showed that the average diameter size of assembled S mRNA-LNPs was 128.66 nm,which was 0.25 times larger compared with blank LNPs,and the potential size was-2.6 ± 0.6 m V,and the encapsulation rate was 97.3%.In addition,the successful expression of FCoV S protein in cells was verified by cellular immunofluorescence(IFA)comparing the difference in S protein expression with bare mRNA transfection and using Western Blot.IFA results showed that the fluorescence intensity of S mRNA-LNPs was higher than that of bare mRNA,indicating that Sn1-mRNA wrapped by LNPs could be effectively protected and delivered into the cell to complete the translation.Finally,the protein expression of LNPs-mRNA was evaluated using a double antibody sandwich ELISA.The results showed that the antigen expression of S mRNA-LNPs was 2.25 times higher than that of naked S mRNA,indicating that the delivery system constructed in this study could effectively protect the target mRNA and improve the translation efficiency.The in vitro transcription and in vitro delivery system of FCoV S protein mRNA constructed in this study can provide a methodological basis for the preparation of FCoV mRNA vaccine and provide a basis for subsequent animal experiments of FCoV mRNA vaccine.The in vitro transcription and delivery system of FCoV S protein mRNA constructed in this study can provide the method basis for the preparation of FCoV mRNA vaccine and the basis for subsequent animal experiments of FCoV mRNA vaccine.