Construction And Identification Of Human Phage Single-chain Antibody Library For Peumoconiosis

BackgroundNow, pneumoconiosis is still the one of the occupational diseases of higher incidence and death ratio, and it's the most serious hazard to the workers. The prevention of pneumoconiosis is still arduous. Pulmonary fibrosis caused by Pneumoconiosis is a long-term chronic process, there is no radical method to eliminate or reversal it up to now, we have no choice but to comprehensively heal according to the illness to relieve symptoms or delay progress. Early diagnosis and active prevent is very important for the the extend of life and the life quality of the patients. The mechanism of pneumoconiosis is still not very clear. In ordor to recognize the occurrence and development of pneumoconiosis, scholars at home and abroad have proposed a variety strategies for research. People did extensive laboratory research and epidemiological studies on the relationship between pneumoconiosis and immunologic function, and found that pneumoconiosis patients all had immunologic function changes. Therefore, it is very significant to find some immne and biochemical indicators toverify if the index can be used as biomarkers of pneumoconiosis.ObjectiveTo construction a human pneumoconiosis phage single-chain antibody (ScFv) library by phage display technology. And then identify its biological activity, expect to provide biomarkers for the early diagnose of pneumoconiosis.MethodsTotal RNA was extracted from the peripheral blood lymphocytes of the pneumoconiosis. Then synthesized first strand cDNA by reverse transcription, VH and VL genes were amplified by semi-nested PCR and were assembled to form ScFv by linker. ScFv and phage vector pCANTAB-5E were digested by Sfi I and Not I respectively with two steps, then we linked them and transformed into competent cells of E.coli TG1,so we constructed the fully human recombinant phage antibody library of pneumoconiosis after super infection by the helper phage. At last, we tested the storage capacity and adequacy ratio of the antibody libraries.ResultsThe results:Formaldehyde denaturing agarose gel electrophoresis shows that 28S and 18S can be seen clearly in the two bands, and the brightness of 28S is approximately two times than 18S. This indicates that the RNA has a better integrity and without degradation.VH fragment is about 370bp, VL fragment was about 320bp, and the assembled ScFv gene fragments (VH-Linke-VL) is about 750bp.And the storage capacity of the phage single-chain antibody library we constructed was 2.4×107. Five monoclonal were extracted randomly, and then digested the monoclonal by SfiⅠand NotⅠrespectively with two steps., positive insertion rate was 100%(5/5).ConclusionVH gene families and VL gene families were successfully amplified. A large human pneumoconiosis antibody library containing 2.4×107 clones was constructed after rescuing the recombinant phagemid from the transformed E.coli TG1 cells. The diversity of the library is very well, and expect providing a new reference for the early diagnosis of pneumoconiosis.