Expression And Purification Of RSV F Protein And Morphological Observation Of Influenza Virus H3N2

Respiratory virus is the main virus that infects the human respiratory system.This disease is widely prevalent in the population,causing cough,headache,fatigue,severe lung disease and other related symptoms,and even death.Therefore,it has attracted widespread attention worldwide.Human respiratory syncytial virus（HRSV）in the elderly and infants and young children with lower immunity,such as the crowd spread widely,causing respiratory infections,causing diseases such as pneumonia and bronchitis,brought huge losses,two kinds of structure of RSV protein F and G protein mediated the combination of virus and host cell,the virus can enter the cell.RSV F protein also has strong immunogenicity,so RSV F protein is considered as an important target in vaccine and drug research and development.The HA protein in influenza viruses,which have caused several pandemics and killed tens of millions of people around the world,mediates the fusion of the virus with the cell membrane,which allows the virus to enter the cell to replicate.RSV virus genome encoding 11 kinds of proteins,which encode the F protein is now popular target protein,which interact with a variety of a variety of proteins in the human body,the original form of the F protein is known as the F₀,F₀protein subunits was cut up to produce F₂,and F₁fusion as a unit,the unit three together form a trimer,At this point,the F protein is functioning normally.In this experiment,F protein gene was synthesized for point mutation and secreted and expressed in HEK293E and Expi293F.After concentration by ultrafiltration,it was carried out affinity chromatography with Ni-NTA.Stable and uniform proteins were obtained by molecular sieve Superose ^TM6 Increase 10/300 GL gel chromatography for negative staining by electron microscopy.Subsequently,proteins that interact with them could be prepared by using similar preparation methods and then combined,and the results were observed under frozen electron microscopy for further analysis.The results of this study lay a solid foundation for the subsequent research on antigenicity and immunogenicity of virus F protein.Most flu vaccines in chicken eggs for matrix as a preparation for production,however,chicken embryos with quantitative restrictions and pollution in the process of production is likely to occur,so the cells as matrix of flu vaccine also gradually began to develop,the MDCK cells is considered sensitive cells of the influenza virus,there are for substrates by MDCK cells of flu vaccine.In different substrates,the HA protein of influenza virus may be different in structure and quantity,and HA protein plays an important role in the infection process of influenza virus.Whether different structure of HA protein will affect the effect of vaccine is still needed to be further explored.In this experiment,we respectively in chicken embryos and MDCK cells proliferation H3N2virus,and the filtered disease venom sucrose density gradient centrifugation and purification,again after PCR detection of the viral nucleic acid in negative dyeing observation by electron microscope,found that by chicken embryos amplification of viral particles and by MDCK cells proliferation of viral particles size and the shape is slightly different,The purified virus particles amplified from chicken embryos were clear,while the purified virus particles amplified from cells had a more chaotic background.The experiment still needed to proceed to the next step,and the differences between the two were carefully observed under cryonic electron microscopy with higher resolution to further analyze the differences caused by different structures.