Screening,Identification And Expression Of Human Phage Antibody Against Recombinant AntigenB Of Echinococcus Granulosis

Objective In order to study immunological therapy, guidance system, targeting drugs against hydatid disease, human single chain fragment of variable antibody (ScFv) against recombinant antigen B (rEgB) of Echinococcous granulosus (E.g) was screened from a phage antibody library by using phage display technology, then it was characterized and expressed in pronucleus system. Methods Recombinant pMal-EgB-JM109 was expressed as fusion protein rEgB-MBP. Factor Xa Protease was used to digest the fusion protein. Flowing through the amylose resin column and hydroxyapatite column, rEgB was purified by the method of affinity chromatography. The semi-synthetic phage antibody library was panned by rEgB, which was coated in a microtiter plate. After 5 rounds of biopanning, positive clones specific to rEgB with good immune activity were demonstrated. After demonstrating the specificity of ScFv by ELISA, PCR, Sfil/NotI enzyme digestion, the gene sequence of ScFv was decided. Then it was sub-cloned into pCANTABSE, the expressive carrier, and soluble ScFv was expressed when IPTG was added under 30癈 for 30h in pronucleus system XL 1-Blue. The protein weight was detected by SDS-PAGE, its immune activity by ELISA, and its specificity to rEgB by-3-Western blotting. Results 1. Factor Xa Protease can cut the fusion protein rEgB-MBP in the correct site and rEgB can be purified by the amylose resin column and hydroxyapatite column.2. One positive clone was selected with both the standard construction and the specific combination capacity with rEgB. 3. The DNA sequence data show that the ScFv gene was composed of 768 bp and the expression product had 255 amino acids. Its code in GenBank is AF407670. 4. Molecular weight of the antibody protein expressed in XLl-Blur is 28 kDa. 5. Western blotting proved its specificity.6. Optimal expressive condition was in IPTG (0.2mmol/L) under 30癈 for 30h. Conclusion ScFv against rEgB of Echinococcus granulosus has been firstly produced and identified by the phage display technology.