| Background and objectiveA large number of studies show that pneumoconiosis is closely related to the immune function, including nonspecific immunity, cellular immunity and humoral immunity. Silica dust, as a kind of semi antigen, can selectively absorb substances forming compound antigen. The lipoprotein can become autoantigens, which were released by the Macrophages in the process of phagocytosis and digestion of silica dust. Therefore, the lung tissue of pneumoconiosis has complex antigenic components. Scholars at home and abroad have observed that the humoral immune function of patients with silicosis was significantly enhancement, but the cellular immune function tended to decrease. Previous studies showed that the workers exposed to dust, silicosis patients exists in pathological conditions appear the antibody, but due to technical limitations, only on one or some antibodies were detected or antibodies to the stages in a certain period and silicosis were discussed. It is lack of the study about antibody changes of full spectrum, the whole and systematic study.As the development of cellular immunology and molecular immunology technology, antibody library technology is more and more mature. It is a new method which is used to investigate silicosis antibody in the development of silicosis. Phage display technology is to phage coat protein P Ⅲor PⅧgene as the carrier and the target gene fragments are inserted into the Phage genome which does not affect the ability of phage infection and exogenous gene fragments were expressed in phage surface. Since Phage random peptide library was established by fusion of random short peptides with the surface protein P of filamentous phage. ⅢPhage display technology has been quite mature. The human pneumoconiosis phage antibody library has been successfully constructed by our task group and some specific antibodies were screened. The expression of these specific antibodies were tested and verified in the blood of workers exposed to dust and the patients of silicosis. Because there are many confounding factors and and immune reaction of the population is more complex in the progress of construction of phage single chain antibody library of human pneumoconiosis. So the construction of experimental silicosis of phage antibody library and screening silicosis related antibody is important. Methods1. The construction of the models of experimental silicosis. We intermittently poured 1 ml of silica suspension into the lungs of rats by injection of non-exposure bronchus, observing the active state of rats and stroking the rats’ chest so that dust dispersed to the whole lungs to lift the asphyxia. The control group was treated with the same method to pour 1ml of physiological saline into the lungs of rats. The rats were executed separately after 3, 6, 9, 12 week and tissue samples and peripheral blood samples were collected.2. Construction of sc Fv gene. We extracted the peripheral blood of the models of experimental silicosis to extract peripheral blood lymphocyte by rat peripheral blood lymphocyte separation. We extracted the total RNA from mononuclear cell and cDNA was synthesized by reverse transcription kit. Specifically, antibody V-domain coding regions are amplified by PCR. Finally, the variable regions were connected to single chain antibody by the linker.3. Construction of phage single-chain antibody library. The single chain antibody and phage particle vector Pcantab-5e were digested by two restriction enzyme and jointed. Recombinant plasmids were transfected into feeling E.coli TG1 with the helper phage super infection. In the end, we constructed the phage single-chain antibody library.4. Enrichment and screening of phage antibody library. The lung tissue protein was extracted from rats of the models of experimental silicosis and the control group. The phage antibody library against the lung tissue protein of the model rats were enriched and screened with the lung tissue protein of the model rats and the group rats for four rounds and the library capacity was detected.5. Specificity detection of ScFvs. 20 single clones were randomly picked and the plasmid were extracted. Plasmids were coated ELISA board and detected OD450 of model(P) and control(N) respectively. Positive clone was P/N>2.6. The identification of positive clones. Positive cloning plasmids were sent to Shanghai Biological Engineering Company to be sequenced. The sequencing resμlts were blast in NCBI website and the sequencing results were translated into the amino acid sequence of a protein and the translation resμlts are analyzed. Resμlts1. We successfully constructed a phage single-chain antibody library of the models of experimental silicosis by phage display technology and the library capacity was 7.2 ×109cfu/L.2. The phage antibody library against the lung tissue protein of the model rat was enriched and screened with the lung tissue protein of the model rat and the group rat for four rounds. We selected three kinds of relative antibody about silicosis by ELISA, respectively anti-IFN gamma, anti-IL-2R and anti ribosomal P protein of single chain antibodies. ConclusionWe constructed a phage single-chain antibody library of the models of experimental silicosis by phage display technology and genetic engineering technology. The library capacity was 7.2 ×109cfu/L. Results show that there may be some specific antigens in silicosis rats. We selected three kinds of relative antibody about silicosis by ELISA, respectively anti-IFN gamma, anti-IL-2R and anti-ribosomal P protein of single chain antibodies. |
PDF Full Text Request