Large capacity of human liver cancer ribosome display single-chain antibody library
Construction of human liver cancer natural sink capacity, diversity good ribosome display single-chain antibody library, to lay a good experimental basis for the development of therapeutic human antibodies. Methods: The collection of 40 cases of fresh human peripheral blood 2mL (including healthy adults and patients with hepatocellular carcinoma), lymphocyte separation and total RNA was extracted by the design of appropriate optimization of primers by RT-PCR amplification of human antibody heavy chain variable region gene (variable region of heavy chain, VH) and light chain variable region gene (variable region of light chain VL) and intergenic regions of the light chain constant region gene (consistregion of the light the chain, CK). Adoption of improved overlap extension PCR (SOE PCR) technology The VH and VL spliced connection peptide Linker (Gly4Ser) 3. After the introduction of building a ribosome display single-chain antibody (scFv) library template the necessary components, including a T7 promoter, a ribosome binding site (Kozak sequence) and the translation initiation codon, as well as the intergenic regions of the CK gene. Template gene fragments Connection T-vector was transformed into E.coli DH5a E. coli colonies identified by PCR analysis and sequencing, constructed of single-chain antibody library. Results: ① Construction of the storage capacity of 2.65 × 1013 human hepatocarcinoma single chain antibody library; ② sequence of positive recombinants by colony PCR and sequencing analysis, we found that the antibody library has a rich diversity. Conclusion: The use of improved overlap extension PCR (SOE-PCR) method, building a high storage capacity, diversity, good human-derived liver cancer ribosome display single-chain antibody library, improve the efficiency of the library building. Antibody library was constructed successfully, and lay a solid experimental basis for the follow-up screening antibodies, antibody modification. At present, liver cancer is second only to gastric cancer, the third most common malignant tumor of the esophagus, the initial symptoms are not obvious, its high relapse rate after treatment is one of the worst prognosis of malignant tumors. Therefore, we constructed ribosome display single-chain antibody library derived from liver cancer patients and normal peripheral blood lymphocytes of the heavy chain, light chain variable region gene, and identification for the further screening of hepatocellular carcinoma-specific antigen single-chain The antibody has laid the experimental basis for the further development of anticancer drugs such as immunotoxins, immunoliposomes, as well as tumor vaccine development have very significant practical significance.