Screening For The Variable Region Of HBsAb By Single Cell And Transcriptome Analysis Of PBMCs After Hepatitis B Vaccine Immunization

Objective: To clone the variable region sequence of hepatitis B virus surface antibody(HBs Ab), research on human immunological responses to HBV vaccination, and provide the foundation for screening monoclonal antibodies for HBV and developing more efficient means of preventing and treating chronic hepatitis B.Methods:(1) A healthy volunteer who is HBs Ab positive was injected with 20 μg of hepatitis B vaccine. Seven days later, his serum HBs Ab titer was greater than 1000 m IU/ml. Thirty ml of heparin anticoagulant blood were obtained for sorting CD19+/CD27+/Ig G+ cells by flow cytometry. And 96 single cells were picked from CD19+/CD27+/Ig G+ positive cells and lysated into 20 μl lysis buffer for single cell. Reverse transcript PCR was used to obtain the variable region of heavy and light chain of each single cell with mixed primers. PCR products were purified and cloned into p MD®19-T vector. Six clones were picked and sequenced. The similarity and homology of sequence were analyzed with DNA Star 5.0 Meg Align and Ig BLAST softwares.(2) Peripheral blood mononuclear cell(PBMC) samples were separated from three volunteers before and after HBV vaccination. The total RNA was isolated from PBMCs. The quality of RNA was confirmed and then used for Gene Expression Profiling analysis. Clean data were obtained from original sequencing results, and then used for differential expression gene analysis, Gene Ontology(GO) enrichment analysis and KEGG pathway analysis.Results:(1) Twelve positive plasma cells were screened. And they showed high similarity and homology in the variable region of heavy and light chain.(2) 829 differential expression genes were obtained, on which an analysis of GO term enrichment was made, and we found that the gene are mainly involved in biological processes like the fatty-acyl-Co A reductase(alcohol-forming) activity, iron ion transpor, protein complex binding and ferrous iron transmembrane transporter activity. The analysis of KEGG pathway showed that the gene are mainly involved in Toll-like receptor signaling pathway, TNF signaling pathway, Cytokine-cytokine receptor interaction and NF-Kappa B signaling pathway.Conclutions:(1) The variable region sequence of the heavy chain and light chain of HBs Ab may be acquired.(2) Important genes and regulatory pathways that may participate in the course of immune responses to hepatitis B vaccine are preliminarily obtained.