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Construction and Preliminary Identification of Immune Phage Display Antibody Library Against Respiratory Syncytial Virus

Author: WangZhiHua
Tutor: ZhangGuoCheng；XuDongLiang；SunXin
School: Fourth Military Medical University
Course: Pediatrics
Keywords: Antibody library Phage display technology Fab antibody Respiratory syncytial virus Respiratory tract infections Child
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 82
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Abstract

[Background] respiratory virus infection is common pediatric diseases, mainly including the respiratory syncytial virus (respiratory syncytial virus, RSV), adeno-virus (ADV), influenza virus type A and type B (IV-A, IV-B), parainfluenza virus (PIV), human metapneumovirus (hMPV), RSV is the most common virus causing respiratory tract infections in children, and 90% in the first 2 years are infected with RSV, serious impact on children's health. Diagnosis, treatment and prevention of RSV infection has been a pediatric academic concern, but so far there is no ideal antiviral and effective RSV vaccine. In 1891, Emil von Behring for the first time the diphtheria antitoxin serum from the animals used in the treatment of diphtheria infection in children, to obtain more satisfactory results, which created the antibody treatment of infectious diseases, the first of its kind in the history of medicine. 1975, hybridoma monoclonal antibody (Monoclonal antibody, McAb) the creation of technology, to promote the rapid development of the Academy of Pediatrics and many other disciplines in clinical medicine, McAb is widely used in the diagnosis of infection, cancer and other diseases, treatment and prevention, shows a good prospect; fly in the ointment is that this murine monoclonal antibody used in the human body produces human anti-mouse antibody (human anti-mouse antibody, HAMA), it only makes treatment with McAb quickly lost utility, or even cause severe allergic reactions and other side effects, its clinical application is very limited, therefore, the murine McAb humanized, and the development of the whole person endogenous antibody goal pursued by scientists from various countries have been. With the development of molecular biology and immunology, the emergence of phage display technology, a new way for the preparation of a variety of different immune the Hominid-derived antibody, the people derived antibodies become possible. 【Objective】 The purpose of this study is to use recombinant DNA technology and phage display technology to build a pediatric respiratory syncytial virus infection in patients with immune phage antibody library against respiratory syncytial virus monoclonal antibody, the antibody library screening and for further study of its biological activity, technology platform to build a human antibody prepared, for children with respiratory syncytial virus infection in the pathogenesis, diagnosis, treatment and prevention to provide a new and effective means. 【Methods】 collected 52 cases of respiratory syncytial virus infection in children in the outer periphery of 2 ml blood of about 100 ml peripheral blood lymphocytes with lymphocyte separation medium separation, extraction of total RNA using the guanidine isothiocyanate / phenol method as a template reverse transcription into cDNA. The longer cDNA as a template, the diversity of antibody kappa light chain gene and the antibody heavy chain Fd gene (Fd segment gene by a heavy chain variable region and first constant region gene) was amplified by PCR. The κ light chain gene amplification product is recovered after Sac I Xba I digested vector fragment will be recovered by the same digestion pComb3x connected with T4DNA ligase and then electroporated into E. coli XL1-Blue competent bacteria to construct the kappa light chain gene library, the transformed bacterium expand After cultivation plasmid was extracted by the Spe I Xho I digested, recycling recovered after the carrier of the large fragment with the diversity of the κ light chain gene by the same double digestion FD segments of the heavy chain gene fragment connected electrically transform competent E.coli XL1-Blue, to build into the Fab antibody gene library. Respectively, with the restriction endonuclease enzyme Sac I Xba I and Spe I Xho I combination double digestion kappa light chain antibody gene library and Fab phage antibody gene library reorganization. Helper phage M13K07 super-infection transformation of the host strain XL1-Blue, constructed Fab phage antibody library and affinity enrichment screening technology to its preliminary screening identification. [Results] good quality total RNA was extracted using the guanidine isothiocyanate / phenol method diversity kappa light chain genes and heavy chain Fd gene was amplified by RT-PCR and by PCR for the desired The target fragment. Last successful build pediatric respiratory syncytial virus infection in patients with human-derived immune phage Fab antibody library, this antibody library containing 2.6 × 106 clones, restriction enzyme digestion with restriction enzymes, of which 70% contain kappa light chain gene and The heavy chain Fd gene phage antibody library constructed storage capacity of 1.8 × 106, the titer approximately 1.06 × 1012pfu/ml, preliminary screening, antibody library has been varying degrees of enrichment. Monoclonal antibodies and their immune activity research has laid a good foundation for the further use of affinity enrichment screening technology, human anti-respiratory syncytial virus. [Conclusion] laid the foundation for the use of recombinant DNA technology and phage display technology, successfully constructed a human monoclonal antibody to create good conditions for the development of safe and effective diagnosis, treatment and prevention of respiratory syncytial virus infection genetically engineered drugs .

Construction and Preliminary Identification of Immune Phage Display Antibody Library Against Respiratory Syncytial Virus

Abstract

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