Cloning The Genes Of H3N2 Subtype Influenza Virus Adapted On Vero Cell And Analyzing The Associated Mutation Sites

At the end of 20th century,the development of the whole virus gene sequencing,the bioinformatics analyzing,proteome and DNA arrays has take a great change on the virus research.Using the bioinformatics database and tools,we can research evolution rules of the influenza virus sequences.By analyzing those sequences,we can forecast the change of virus main antigen sits or the most important residue change of virus adapted new host.This technique gives hope for vaccine development that could not success by the traditional methods.At the same time,it also forecasts the pandemic of virus adapted new host and save a lot of time for alarming.There are many aspects should be promoted of the influenza vaccine production.For example, the vaccine produced by egg embryo cope with influenza pandemic is not enough. Veto cell is WHO resigned cell strain to produce vaccine.It can be cultured at large scale by the bioreactor.It has great foreground to produce influenza vaccine.But Veto cell is not the influenza virus susceptible cell naturally.So it is very important to built genome of the influenza virus adapted on the Veto cell and using the reverse genetics methods to prepare the influenza virus seeds to product the influenza vaccine.In this experiment,I used the influenza virus that adapted on the Vero cell A/kunming/1/2005va and designed the primer contained Esp3I and Eco131I enzyme cutting sits.Using the RT-PCR technique to acquire the A/kunming/1/2005va 8 whole segments,this strain has adapted on the Veto cell.By the sit mutation technique let the two Eco131I sits of PB2 segment disappear.After inserting those 8 segments to PHW2000 vector separately,I get the genome of the influenza adapted on the Veto cell.This gives the gene pool of the influenza that can get the high production on the Veto cell by genetic reassortment.I also compared the A/Kunming/1/2005 and A/kunming/1/2005va NP,HA and NA genes and protein sequence.It serves theory bases of virus evolution.It also gives bases guides to rebuild the influenza for improve the production output and using Vero cell product influenza virus vaccine.