| Autor: |
Zhang Guoli, Tian Yuan, Yu Wang, Li Zehong, Hongyang Man, Sun He, Yue Yuhuan, Wu Guangmou, Jiyuan Zhang, Yue Qiu |
| Rok vydání: |
2021 |
| Předmět: |
|
| DOI: |
10.21203/rs.3.rs-817839/v1 |
| Popis: |
Background: Influenza virus matrix protein M1 is encoded by viral RNA fragment 7 and is the most abundant protein in virus particles. M1 is expressed in the late stages of viral replication and exerts functionality by inhibiting viral transcription. The M1 protein sequence is an attractive target for antibody drugs.Methods: The M1 protein sequence was amplified by RT-PCR using cDNA from the H5N1 virus as a template; the M1 protein was then expressed and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. The differences between HuScFv and TAT-HUScFv were verified by various experiments and the amino acid binding site of the M1 protein was determined.Results: The M1 protein of H5N1, HuScFv, and TAT-HuScFv, were successfully purified and expressed by and in E. coli. Further analysis demonstrated that TAT-HuScFv inhibited the hemagglutination activity of the 300TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.Conclusion: Recombinant HuScFv was successfully identified using the Tomlinson (I+J) phage antibody library and successfully linked to the TAT protein transductive domain of the HIV virus. Compared with the HuScFv, the addition of the TAT peptide improved its ability to penetrate the cell membrane. A definite amino acid binding site was identified after the decomposition of M1 protein, thus providing a target and reference for the development of antibody drugs and the study of new drugs. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
