| Avian influenza caused by influenza A virus does severe harm to human health and livestock industry, and seriously affect the country's economy. Avian influenza virus genome is prone to mutate, resulting in polymorphism and bringing great difficulty to prevention. At present, it is unclear which specific mutations can make the H5N1 virus easily spread in people. If the H5N1 virus has the specific mutation to spread in people, it will cause disaster to human being.Vaccine and antiviral drug are usually employed to prevent and treat avian influenza nowadays. It will take a long time to produce the protective antibodies after the injection of vaccine. The resistance of avian influenza virus to chemotherapy agents (ion channel inhibitor and neuraminidase inhibitor) often influences the disease control effect. In recent years, passive immunization on prevention and treatment of avian influenza is one of hot-spot researches. Antibody products used for passive immunization against avian influenza produces protective effect immediately after administration, which can make up for the lack of vaccine and drug resistance.Avian influenza is a respiratory infectious disease, infected mainly through respiratory and digestive tract, where secretory immunoglobulin A (SIgA) plays an important role. As a passive immune agents, SIgA can be administrated before or in the early stage of infection. In addition, administration of SIgA can be made by the intranasal/aerosol or oral/gavage route, which is not only convenient but also safe. Therefore, it is of great value to study the blocking effect of SIgA against avian influenza infection in respiratory and digestive tract.Secretory IgA antibodies has unique characteristics in the structure and function. Compared with the normal antibody molecules (IgG, IgA), SIgA antibodies have many excellent features. SIgA, which consists of two monomer IgA, has a higher functional affinity. The presence of secretory component (SC) improves the stability of SIgA and enables SIgA to resist acid and protease degradation. A study showed that the SIgA can exist in human exocrine ducts more than four months. In addition, the non-specific activity to pathogenic microorganisms of SC endows the SIgA non-specific immune protection. Moreover, SIgA can adhere to epithelial surface neatly by carbohydrate residues, forming a protective layer to prevent the invasion of the pathogens.Based on the light and heavy chain expression plasmid of anti-H5N1 HA chimeric antibody and human SC, IgJ expression plasmid, which have been constructed in our laboratory, we co-transfected these expression plasmids into the Chinese hamster ovary cells (CHO) and established monoclonal cells stably secreting anti-H5N1 HA SIgA. The methods and results are as following:1. Construction of chimeric SC and IgJ expression plasmidsThe SIgA component genes (SC, IgJ, constant region of human IgA heavy chain, constant region of human Kappa chain) was cloned in our laboratory. Because of changes in the experimental design, we cloned SC, IgJ into the vector pcDNA4/HisA, which uses zeocin as selection marker, to construct the eukaryotic expression plasmids pCDNA4-IgJ, pCDNA4-SC respectively.2. Expression of anti-H5N1 HA SIgA antibody in CHO cells and analysis of its immunological characteristicsCHO/dhfr- cells were co-transfected with plasmids of light and heavy chain of anti-H5N1 antibody, and cell lines expressing of anti-H5N1 monomeric IgA were selected. The immune characteristics of the recombinant IgA were analyzed. To express the secretory IgA antibody, we co-transfected the SC and IgJ expression plasmids into the Chinese hamster ovary cells (CHO) stably expressing monomeric IgA. Then we used Zeocin to select monoclonal cells stably secreting SIgA. The SIgA antibody secreted from the CHO cells was confirmed by Western blotting and ELISA, which demonstrated that the complete SIgA molecules with the ten subunits were produced. ELISA experiments also showed that the SIgA had a good binding capacity with H5N1 HA antigen.3. Improvement of the SIgA expressionMethotrexate (MTX) is the inhibitor of dihydrofolate reductase (DHFR) protein. When there is MTX in the medium, MTX can penetrate into cells and combined with the DHFR protein, blocking the synthesis of nucleotides. Inhibition of DHFR can be exployed to screen cell clones with duplicated DHFR genes which express more DHFR to compensate the inhibition of its activity. Gene of interest is usually duplicated along with the nearby DHFR gene, which results in high level of expression of the target gene.In this study, the concentration of MTX was increased gradually from 5nM, to 25nM, and then to 125nM. First, the cell lines were cultured in medium containing 5nM MTX. When the cells grew to 90% confluence, the cells were passaged with 1:4, and this dividing procedure was repeated five times before 25nM MTX was used. Similar procedures were carried out to screen cell clone with 125nM mTX. Finally, high level expression cell lines were screened with serial dilution method.4. Fermentation of SIgASIgA monoclonal cells were cultured in serum-free medium (CD CHO medium with 125nM MTX) and the cells were domesticated for 2 months, with the concentrations of DMEM and dialysis serum was decreased gradually until the cell was fully adapted to CD CHO medium. The adapted cell lines were subjected to suspension culture for 2-4 weeks in CD CHO medium (with 125nM MTX), and then the cell culture supernatant (1L) was collected to be SIgA purification by Protein-L affinity chromatography.5. Preparation of monoclonal antibodies against SCFor detecting SIgA, hybridoma cell lines secreting monoclonal antibodies against human SC were developed. The SC prokaryotic expression vector pQE80L-SC was constructed and the expression of SC was induced in E.coli. Mice was immunized with the purified recombinant SC, and two monoclonal antibodies (AB07, EC11) were selected. These two monoclonal antibodies were used to establish a rapid assay for SIgA detection. |
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