Cloning, Expression And Analysis Of Mitochondria Antiviral Signaling Protein And The Preparation Of Monoclonal Antibody

The innate immunity system is the first defensive line for the host to resist exogenous microbial invasion. After the innate immune response are activated, immune protein factors will transmit signals step by step, and then produce inflammation and immune reaction to remove most of the pathogens. Mitochondria antiviral signaling protein (MAVS) participated in the antiviral immune signal transmission, and play a critical role in the process of the body’s immune defense.In this study, a full-length open-reading frame (ORF) of Human MAVS (HMAVS, GenBank accession number:DQ174270.1) was amplified from 293T cells, inserted into pGEX-4T-2, and transformed into Escherichia coli, which was then induced to express the foreign gene. However, no recombinant protein was detected by using SDS-PAGE and Western blotting analysis, which might be caused by the codon preference. Subsequently, the immunogenicity of the polypeptide sequence encoded by the ORF of HMAVS was analyzed, which showed that the latter part (295-540AA) of the polypeptide was of higher immunogenicity. Therefore, a truncated gene fragment (883-1623bp) encoding the 295-540AA was amplified and designated as HMAVSt. HMAVSt was inserted into a bacterial expression vector pGEX-4T-1. Then, the recombinant plasmid was transformed to E. coli, and the expression of recombinant proteins was induced by the addition of Isopropyl β-D-1-Thiogalactopyranoside. SDS-PAGE result showed that E. coli successfully expressed fusion protein rHMAVSt (GST-HMAVSt) with a molecular mass of 53.7 kDa, which were consistent with the theoretically predicted molecular weight. The rHMAVSt was used to immunized BALB/c mice. The murine antisera was used in western blotting to analyze 293T cell lysate, the result showed that the antisera against rHMAVSt recognized the native HMAVS protein from 293T cells. It showed that rHMAVSt protein had good immunogenicity, which could be used to replace native HMAVS for antibody production. And polyclonal antibody against rHMAVSt was successfully achieved. The spleen cells from rHMAVSt immunized BALB/c mice were extracted to fuse with myeloma cells. After eradicating non-fused cells, an enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test (IFAT) were used to screen for the positive colonies, and the colonizations with limiting dilution method were carried out 3 times. At last, positive hybridoma cell lines expressed anti-rHMAVSt antibody at high level were obtained successfully. The cells was injected into the abdominal cavity of BALB/c mice for the production of monoclonal antibody ascites, IgG subtypes of which was then detected. HMAVS sequence was also inserted into eukaryotic expression vector pcDNA3.1/HisA and transmitted to 293T cells, the over expression of HMAVS was detected in 293T cells by using western blotting analysis. The monoclonal antibody and the recombinant pcDNA3.1/HisA/HMAVS provide important materials for further study on the mechanism of MAVS proteins in human antiviral immune signaling pathways.