| The influenza virus is currently one of the main factors threatening human health.Influenza virus happened in recent years, such as H7N9, H5N1 and H1N1 avian influenza,can cause patient morbidity, high mortality, and it has serious impact on human health, economic development and social stabilityit. Now the main way to deal with the influenza virus are vaccines and drugs. Vaccine is one of the most effective way to prevent influenza virus infection, but in the influenza large-scale outbreak period, the best way to control disease is drug therapy. But the vaccine cannot control the new subtype influenza strains. Antiviral drugs has treatment effectbefore the new virus resistant strainsappear, but the treatment effect is not obvious in late infection. The anti- influenza drug M2 ion channel protein inhibitor and neuraminidase(NA) inhibitor, Such as Oseltamivir and Amantadine can effectively control the condition. But with medication, it prones to drug-resistant strains,and medication must also be in the period of effective drugs,or treatment effect was significantly reduced. There was the use of recovered patient's sera to severe influenza clinical treatment. But getting high-affinity neutralizing antibody from recovered patient's blood is difficult to deal with outbreak of influenza after all, and acquired high antibody titer.This research uses rhesus monkeys, who has high degree of homology in the genetic with human. Rhesus monkeys will produce specific neutralizing antibodies after immune to influenza virus vaccine several times. Through multiple immune to stimulate B cells, it promotes the generation of high affinity antibodies.Using FACS to sort B cellmarking CD3-/CD19-/CD27-/CD80++. Then using the single cell PCR technology to clone antibody gene, and express in 293 T cells. So we can get antibody gene, whose variable region ofheavy chain and light chain can naturally match.Using single cell PCR to clone antibody gene is mainly used in plasma cell and memory B cell clone.Compared with the traditional method of antibody production, single cell PCR technology have more advantages in high efficiency and total anthropogenic, genetic diversity. Finally, using ELISA method to detect antigen-antibody affinity, BLI technology for quantitiveaffinity analysis, and virus micro-neutralizationexperiment for testing neutralization titer of antibody and so on.This research screened a strainof high affinity neutralizing antibody named 2G11.using the experimental method above. Results in vitro analysis show that: ELISA and western blot results show that, 2G11 can combine H7N9-Anhui strain. And compared with the reported broadly neutralizing antibody named FI6, the binding activity of 2G11 is 1000 times than FI6. virus micro-neutralizationexperiment also prove that 2 G11 can neutralize H7N9 influenza virus well. The experimental data show that, 77 ?g/ml supernatant of antibody can still neutralize 90% of the virus after diluted in proportion of 1:320. And its neutralizing activity is much higher than reported antibody FI6. Biological membrane layer interference(BLI) experimental results also show that 2G11 has a strong affinity to antigen.Its affinityreaches level 0.6 n M. Comparing to other reported antibodies, its affinity level is pretty high. HI experiment also proves that 2G11 has a very high neutralization degree.This research screened a high-affinity neutralizing antibody named 2G11 using single cell PCR technology from immune rhesus monkeys,which can specifically neutralize H7N9 virus. It will provide effective basis and thinking to clinical treatment of highly pathogenic influenza virus. |
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