Construction And Preliminary Identification Of Immune Phage Display Antibody Library Against Respiratory Syncytial Virus

【Background】Respiratory syncytial virus(RSV) infection is very common in children. RSV is a major etiological agent of lower respiratory tract infection in infants. During the RSV season, it is estimated that about 40% of children will develop a lower respiratory tract infection. Outbreaks occur annually, and attack rates are high; 60% of children are infected in the first year of life, and 90% by age 2 years. The diagnosis, therapy and prevention of respiratory syncytial virus infection are always the focus of pediatrician and scientists. But the effects of various available antiviral drugs are usually not completely satisfactory. In 1891, Emil von Behring cured a little girl of her diphtheria using antitoxinum diphthericum. It was the first case in history using antibody as drug for infection diseases and laid a foundation for the development of monoclonal antibodies based infection diseases therapy. In 1975,the invention of hybridoma technology that allowed production of monoclonal antibody(McAb) facilitated the development of pediatrics and other subjects rapidly. Monoclonal antibodies(McAbs) revealed favorable prospect when they were widely used in the diagnosis, therapy and prevention. But for human therapy, the murine McAbs were limited greatly because of the human anti-mouse antibody (HAMA) response and other unexpected side effects. With the development of molecular biology, the advent of genetic engineered antibody technology, particularly the phage antibody library technology provided a good and new approach to preparation all kinds of humanized or human McAbs and it became a revolutionary progress in antibody engineering field.【Objective】To construct a human phage display antibody library, which will help to develop new drugs and vaccines against respiratory syncytial viruses and solve many of the issues that have limited the progression and application of murine antibodies in the clinic, provide a platform for human antibody preparation and new diagnosis, prophylaxis and therapy for the children with respiratory syncytial virus infection.【Methods】Peripheral blood lymphocytes were isolated from 100ml blood, which was obtained from 52 children with RSV infection. Then the total RNA was extracted immediately by Trizol and trichloromethane. Meanwhile, the integrity of RNA was checked by alkaline denaturing agarose gel electrophoresis and optical density (OD) measurement. A set of PCR degenerate primers forκlight chain and heavy chain Fd genes of antibody were used according to the reference. The cDNA was synthesized from the total RNA of lymphocytes according to the manual of RNA reverse transcription kit and used as templates for the followed PCR performance. The amplified light chain gene products were digested with restriction endonucleases Sac I + Xba I and inserted into phagemid vector pComb3x and the clone samples were electroporated into competent E.coli XL1-Blue to yield recombinant phage antibody gene library ofκlight chains. The transformation efficiency was detected to calculate the capacity of light chain gene library. Ten positive colonies were picked up randomly for the restriction enzyme analysis by Sac I + Xba I to identify recombinant rate. To select the macromolecule fragments, the plasmids were extracted from all the positive colonies and cut with endonucleases Xho I + Spe I. The macromolecule fragments and Fd fragments cut with the same endonucleases were ligated by T4 ligase. The ligation products were electroporated into competent E.coli XL1-Blue to yield recombinant phage antibody gene library of Fab. The next steps were similar to the above. The final transformed cells were infected with M13K07 helper phage to yield recombinant phage antibody of Fabs.【Results】By recombination of light chain and heavy chain genes, using 100ml peripheral blood lymphocytes, which was obtained from 52 children with respiratory syncytial virus infection as gene sources, an immune phage display antibody library against respiratory syncytial virus containing 2.6×106 different clones was constructed successfully, in which 70 percent clones hadκlight chains and heavy chain Fd genes. The capacity of Fab phage antibody gene library was 1.8×106 and the titre of the original Fab antibody library was about 1.06×1012pfu/ml. The antibody library gained enrichment in different degree after the preliminary screening.【Conclusion】Utilizing the technology of phage display, an immune Fab phage display antibody library against respiratory syncytial virus was constructed successfully in our study, which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs, and may also contribute to the improvement in the diacrisis, therapy and prophylaxis of respiratory syncytial virus infection in children.