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Construction of the Primary Phage Display Single Chain Antibody Library Against Human Hepatic Fibrosis

Author: WuHaiEn
Tutor: ShuJianChang
School: Jinan University
Course: Internal Medicine
Keywords: Liver fibrosis Phage antibody library Single-chain antibody
CLC: R575.2
Type: Master's thesis
Year: 2008
Downloads: 51
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Abstract

The purpose of building large storage capacity, the specificity of the anti-human liver fibrosis primary phage antibody libraries, scouring lay the foundation for further screening of antibody libraries to provide new ideas, new methods for early diagnosis and treatment of liver fibrosis. The method of using human hepatic stellate cell line LX-2 intraperitoneal injection of immune Balb / c mice, mouse serum antibody titers were detected by ELISA. The spleen was removed under aseptic conditions and ground to a tissue homogenate, application Trizol reagent extracting total RNA electrophoresis and absorbance (OD260/OD280) detection of RNA or other degradation of its purity. The Poly AT tract'mRNA purification system purified mRNA. Determination mRNA content and OD260/OD280 of value. Random hexamers as primers, mRNA as a template, adding the reverse transcriptase, the first strand of the synthetic single-stranded cDNA fragments. The first strand cDNA as a template, PCR amplification of the antibody heavy chain variable region (V_H) and a light chain variable region (V_L). Will equal weight mixed light chain amplification product with linker primers, the the synthesis V_H-linker-V_L, joining primer, at its 5 'end and 3' ends were designed with endonucleases Sfi I and Not I restriction sites point, PCR amplification, and assembled into ScFv gene. ScFv gene was excised by the restriction enzymes Sfi I and Not I digestion, purified and quantitative digestion product. The ScFv gene similarly by Sfi I and Not I-digested phage vector pCANTAB 5E connection formed ScFv / pCANTAB 5E gene fragment with T4 DNA ligase. The electroporation method would ScFv / pCANTAB 5E transformed into E.coliTG1 competent cells, painted SOBAG tablet, adding helper phage M13K07 save, Construction of phage antibody primary library. Anti-human liver fibrosis primary phage antibody libraries were identified through the following steps: (1) random SOBAG tablet picked three colonies, after extraction of phage amplification, agarose gel electrophoresis extract; (2) extracted phage as a template, PCR amplification with RS primer electrophoresis observation amplification product; ③ will extract the phage with restriction enzymes Sfi I and Not I digestion, electrophoresis after digestion product. Results mouse anti-human liver fibrosis antibody titer of 1:512. After electrophoresis of the total RNA extracted three bands, suggesting no degradation and OD260/OD280 value of 1.95, the concentration of the purified mRNA was measured obtaining 253ug/mL, OD260/OD280 value 2.03. By RT-PCR the successfully amplified heavy chain (V_H) and light chain (V_L), the size of the gene fragment is located between 250bp and 500bp. Light and heavy chain gene by overlap extension PCR series, the single chain antibody gene size of about 750 bp. ScFv / pCANTAB 5E the ScFv gene phage vector pCANTAB 5E connection formed gene fragment was transformed by electroporation into E.coli TG1 competent cells within the primary repository of the anti-human the liver fibrosis phage single-chain antibody successfully constructed, the capacity of the antibody library 6.5 × 10 ~ 6. Colonies extracted in the self-similar to the size of the associated phage phage amplification. Per amplification and endonuclease digestion, agarose gel electrophoresis are visible the ScFv fragment of about 750bp. Conclusion The successful construction of the large storage capacity of the anti-human liver fibrosis primary phage antibody library.

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