Preparation Of CDNA Library From Hybridoma Cells With Monoclonal Antibody For Influenza B Virus And Bioinformatics Analysis

Influenza caused by influenza virus was a acute respiratory tract infectious disease, spread rapidly and easily antigen variation. Virus′s infection process is actually the process that mutual struggle process of virus and the host or organism. hybridoma cell was a special composite cell,whose secretory antibody gene was originated B cell which was a immunocyte irritated by immunogen, B cell′s genome bore the weight of interactive information between infector and parasitifer, meanwhile, integrated with macrobiotic gene originated by tumour cell, so hybridoma cell had a relative survival time, maybe mutation and recombination were generated, then produce material besides antibody. Researching of genome of hybridoma cell was a Subject worth exploring. How to obtain biological information fast and efficiently from the genome has become an urgent and challenging task, EST technology was just developed based on this understanding.Influenza B virus was used for the immunization of BALB/c mice. The hybridoma cell strains stably secreting McAbs against Influenza B virus were obtained by fusion of murine splenocytes with SP2/0 plasmacytoma cells, and the secreted McAb was identified. The hybridoma cell was cultured in vitro. Total RNA of hybridoma cell was extracted from the cultured cells and then mRNA was extracted further. Moreover ,single - strand cDNA and double - strand cDNA were synthesized in turn. The double - strand cDNAs were ligated to EcoRI adaptor ,which were later ligated to arms of pBluescriptⅡSK(+) XR.Ligated - cDNAs were packed in vitro, then the hybridoma cell cDNA library was constructed. Sequence of EST was obtained by sequencing random, then had BLAST index through Unigene Server of National Center Biotechnology Information(NCBI). The sequencher software was used to set up and analysis EST contig. In order to establish the foundations that studing gene and predict or exploitation function of hybridoma cell.The results show that: 1) Three hybridoma cell strains which can steadily secrete specific McAbs were obtained, named 3B2,12G3,5A4. chromosome number of hybridoma approach that of parent cell, McAb heavy chain belonged to IgG1, and light chain belonged to Kappa. titer of McAb secreted by three strains was higher ennogh. The specificity identify showed that McAb did not react with influenza A and RSV. 2) The hybridoma cell cDNA library consisted of 1.27×106 recombinants with the length of 0.5～2.0kb and the recombinating efficiency was 90.63% . The amplified library was 3 .5×107 recombinants/μl in concentration and the number of negative colony was the most suitable in density after it was diluted to 10 - 6 in concentration. 3) 400 clones were selected randomly from archaelibrary to sequence, 384 sequences were obtained from the plamids, and 320 ESTs were proved to be useful after edited using biosoftware. After analysis by using the BLAST program, 118 ESTs were proved to be the known-genes, and ESTs whose aligment value were low maybe unknown-genes. Contig analisis of 320 ESTs by Sequencher 4.8 showed that 25 comtig including 71 ESTs which was 22.2% in all EST were obtained. The rest EST were single-copy sequence.Conclusion: 1) The McAbs established by our method could be highly specific for Influenza B virus, which provides a basic condition for development of rapid diagnosis method for Influenza B virus. 2) The constructed cDNA library of hybridoma cell and sequencing of partial clones provided useful resource to study function of new genes, further detecting target genes, at the same time, to predict or exploit function of hybridoma cel. 3)Accomplishment of this thesis is a important initial work for building resource information databank of hybridoma cell.