Establishing a genebank for phage single-chain antibody using silicotic rats / 中国职业医学

ABSTRACT

OBJECTIVE: To establish a genebank for phage single-chain antibody for further screening the specificity of single chain fragment variable( Sc Fv) in lung tissue of silicosis rats by phage display technology. METHODS: Twenty-four specific pathogen free male SD rats were used to construct silicosis model by one-time bronchial perfusion with 1. 0 m L of silicon dioxide suspension( mass concentration,100 g / L). We took periphery blood from 6 rats 3,6,9 and 12 weeks respectively after establishing the model. The peripheral lymphocytes were mixed,and total RNA was extracted using Trizol,and c DNA was synthesized by reverse transcription. The degenerated primers were used to amplify the variable region of heavy chain( VH) gene and variable region of light chain( VL) gene by polymerase chain reaction( PCR).Then VH and VL genes were assembled to form Sc Fv by T4 DNA linker. The cloning recombinant of Sc Fv and plasmid of PCANTAB-5e were transformed into competence E. coli TG1 by calcium chloride. The Sc Fv genebank of silicosis model was constructed by M13K07 helper phage superinfection. There were 10 bacterial colonies for plasmid restriction dualenzyme digestion randomly selected for confirmation. RESULTS: Agarose gel electrophoresis showed that there were two bands of obvious 28 S and 18 S in total RNA of periphery blood lymphocytes of silicosis rats. The total RNA was intact. The size of VH gene fragment was about 400 bp,the size of VL gene fragment was about 350 bp and recombinant Sc Fv gene fragment length was about 750 bp. The helper phage was amplified and placed with double-deck agar plate and observed limpid plaque with the size of a rice grain. The phage titer was 1. 35 × 10~(16) pfu / L. The recombinant plasmids were transformed into E. coli TG1 and total bacterial count was 8. 0 × 10~9 cfu / L in resistant plate. The positive cloned plasmid PCR gel electrophoresis and double enzyme results showed a positive inserting rate of 90. 0%. The capacity of phage single-chain antibody genebank of experimental silicosis was 7. 2 × 10~9 cfu / L. CONCLUSION: The silicosis rat model with phage Sc Fv gnebank could be successfully established,and its capacity and diversity provide support for the follow-up screening.