Proof Of Concept Based On Marburg Virus Messenger RNA Vaccine

Marburg virus belongs to filamentous RNA virus.The disease caused by it is called Marburg hemorrhagic fever.It is an acute and highly lethal infections disease mainly manifested by severe bleeding,and is considered as one of the most terrible diseases in the word.There is currently no vaccine or effective treatment against the disease,so it is necessary to accelerate the development of a vaccine against Marburg virus.Glycoprotein(GP),as the only glycoprotein on the surface of Marburg virus,has high antigenicity,and is also the main protein that stimulates the body to produce immunogenicity.Therefore,this protein sequence is selected as the target sequence,and develop a new vaccine for mRNA that can be quickly translated into protein—mRNA vaccine,and its immunogenicity is studied at the animal level.The research contents and results mainly include the following aspects:(1)The mRNA sequence was determined,and the mRNA was packaged by the LNP packaging method.The microfluidic technology was used in the packaging process.The detection items included the encapsulation rate,particle size and zeta potential.(2)Verify the accuracy and reliability of the mRNA concentration measurement using the Nanodrop.Explore the differences in lying EGFP-mRNA-LNP samples in different ways.The experiment results show that different lysis methods have a great impact on mRNA,and the lysis method still needs to be further optimized.(3)In terms of affecting mRNA stability and translation,the length of the poly(A)tail of the mRNA was determined in this project,and the length of the poly(A)tail was determined to be 43bp.This article further verified the accuracy of the MVP-mRNA sequence,reverse-transcribed the MVP-mRNA sample into a DNA sequence,confirmed the correct size by electrophoresis,and the sequencing results were consistent with expectations.(4)The liposome transfection system was used to transfect naked EGFP-mRNA and MVP-mRNA into HEK293 cells.According to the results of transfection,it was seen that naked EGFP-mRNA had fluorescence after 1h.WB results showed that naked MVP-mRNA was able to express MVP,and the feasibility of mRNA design was initially determined;MVP-mRNA-LNP did not produce significant fluorescence.(5)The immunogenicity was evaluated from two parts,immunohistochemistry and ICS detection.The results showed that,under the condition of single-needle immunization,the MVP protein was locally expressed in the muscle site,but not detected in the liver site at 24 hours.ICS tests were performed after the second immunization.Both 10μg and 50μg of immune dose mRNA can cause MVP to produce specific CD4+T cells.Therefore,the MVP-mRNA-LNP vaccine designed in this thesis has good immunogenicity.(6)The pcDNA3.1(+)-MVP plasmid was constructed to express the MVP protein and used as a subsequent ELISA to detect antibody levels.The target fragment was ligated to the pcDNA3.1(+)vector after digestion by PCR.The ligation product was verified by colony PCR and double digestion.The results proved that the pcDNA3.1(+)-MVP recombinant plasmid was successfully constructed.(7)After several experimental explorations,it was finally confirmed that the TMAE anion exchange followed by Cu column affinity chromatography purification method.WB results showed that the MVP protein was successfully expressed and could be used to detect coated antibodies by ELISA.