Anthropogenic Anti Hbsag Immune Function Verification Dsfv Antibody Targeting Interferon

Interferon (IFN-α) and nucleoside analogues, which are commonly used drugs for treatingchronic hepatitis B (CHB) all have antiviral activity and inhibit the DNA replication of hepatitisB virus (HBV).But many problems exist in nucleoside analogues,such as drug resistance,unstable curative effect and safety of long-term use. Therefore, interferon becomes the goodchoice drug for treatment of CHB. However, the plasmic half-time of conventional interferon isshort and needs to use large doses for a long time. Moreover, it is easy to relapse after drugdiscontinuation and cannot even eliminate cccDNA thoroughly. To sum up, it is of greatsignificance to open up new and effective ways to remove the tolerance-inducing original, breakand change over the deep immune tolerance.With the development of science and technology, the antibody technology espands fromthe serum of polyclonal antibody to genetic engineering antibodies. The laboratory member hasscreened a human anti-HBsAg gene engineering antibody from the phage antibody libraryand got disulfide-stabilized dsFvα pr+(disufide-stabilized Fv fragments) antibodies through thetechnology of PCR site-directed mutagenesis. The laboratory assistants connect the light chainand heavy chain gene of human anti-HBs dsFvα pr+IFNα antibody and put them in the sameopen reading frame(ORF) through itself cutting peptide F2A, which would make light chain andheavy chain of antibody tandem express series in the same plasmid. At last we connected it withthe vector pEE14.1and successfully obtain the restructed plasmid pEE14.1-dsFvα pr+oftargeted interferon.This paper will research immune function of the gene antibody and discuss the immunemechanism. We have proved that targeting interferon could significantly improve theperformance by transgenic mouse experiments.First of all, we design animal experiments and construct three groups of control plasmidsby the enzyme cutting and polymerase chain reaction (PCR): plasmid pEE14.1-IFN-α andplasmid pSCK-IFN-α that only expresses human IFN-α gene, plasmid pSCK-dsFvα pr+thatexpressed targeting interferon composite gene. We verify the expression in eukaryoticmammalian cells293T and detect expressive level by ELISA, western and so on. Secondly, thesesix plasmids including： pEE14.1-IFN-α and pSCK-IFN-α, pSCK-dsFvα pr+andpEE14.1-dsFvα pr+, pVAX1-IL12-dsFvα pr+and pSCK-IL12-dsFvα pr+. We immuned thetransgenic mouse of6-8weeks of age at an interval of7days, and totally immuned one times,for each group of3mice, and one mouse was administered30ug. Finally the experiment used theelectroporation technology to deliver the plasmid and explored the optimal electroporationconditions, which is60V,50ms. Before and after each immunization, blood was collected. And virus copy quantity in serum of before and after treatment of transgenic mice could be checkedby the quantitative PCR, which can reflect the effect of drug treatment.The results confirmed that the plasmid could be expressed successfully. The animalexperiments showed that humanized antibody targeting interferon had obvious advantages overordinary interferon and it can significantly reduce the amount of viral. Antibody targetedinterferon connected to the interleukin-12was much better than before. New electroporationcondition expressed high efficiency compared with the180v,25ms, and could reduce theIrritation and damage of the local muscle tissue injection.In summary, the human anti-HBsAg dsFvαantibody targeting interferon aim at theexcessive tolerance-inducing antigens in the bloodstream such as virus particles and HBsAgvirus particles, and it makes the immune therapic preparation with antibody combine targetedwith virus particles which exist in the blood, besides it is helpful for the virus particles to berecognized by the body and cleared by the immune system, and thus greatly remove the immunetolerance.There is huge potential for research and development.