Analysis Of The Tree Shrew Immunoglobulin Heavy Chain Locus And Development Of Methods To Measure Humoral Immune Response

Tree shrew?Tupaia?,belongs to the Scandentia,Tupaiidae family,has been shown high similarity to primates in neurodevelopment,virus infection and physiological anatomy;Compared with primates,tree shrews have the advantages of small size,easy operation and short reproductive cycle,making it an ideal experimental animal for preclinical studies.Therefore,scientists use tree shrews as one of the animal models for studying human diseases.Although tree shrews are widely used in scientific research,the gene locus of the immunoglobulin and the mechanism of the diversity have not been clearly,which lead the lack of corresponding humoral immune detection means.For the reason that,the animal experiment is limited in the level of pathological features,and the research lack of humoral immune response in the specific pathological conditions.So,in view of the lack of detection methods for tree shrews' humoral immunity,through the second-generation sequencing compared with transcripts and analysis the characteristics of antibodies to determine the gene sequence and distribution of heavy immunoglobulin chains in tree shrews,and cloned the immunoglobulin sequence to prepare genetically engineered antibodies.The sequence of IgHC Locus?113.489kb?of tree shrew immunoglobulin heavy chain gene in Scaffold143596₁,but there have 20 gaps.So,the first task is to fill the gaps in the genome sequence,compared with tree shrew's blood transcripts and analysis the site and homology of antibody domain with other antibody,then clone the heavy chain full sequence of tree shrew by RACE technology and finally ensure the immunoglobulin heavy chain locus and the type of antibody.According to the sequence of lamada chains?Gene ID:102495356?and kappa chain?Gene ID:102503246?,clone the tree shrews immunoglobulin light chain sequences and construct plasmid,through CHO expression system to express and purify recombinant immunoglobulin;The purified protein was used as an immunogen to immunize BALB/c mice and goats,obtained the mouse anti-tree IgG and goat anti-tree IgG.Then detect the cross-reaction between antibody and other species serum.Using tetanus toxoid,group C N.meningitidis cap-sular polysaccharide?GCMP?,HSV-1 virus,HCV virus immunization and infection of tree shrews and mice,detect the changes of antibody levels.Above the experiments,the immunoglobulin heavy chain locus of tree shrew was determined,and development of methods to measure humoral immune response,which filled the lack of humoral immune detection means of tree shrew and laid a foundation for the establishment of animal model of tree shrew in the future.