Screening Of Human ScFv Against IL-8 From Phage Antibody Library

Psoriasis is a kind of common chronic inflammatory disease of skin, its cause is complicated, various kinds of internal and external factors participate in its pathogenesis. The immune system of psoriasis is abnormal, many cytokines act as main effect molecules in the pathology of psoriasis.IL-8 is one of the main abnormally expressed cytokines in psoriasis. Keratinocytes, monocytes, macrophage and endothelial cells are the main sources of IL-8 in psoriasis patients. While IL-8 receptors are distributed in keratinocytes, neutrophils, T cells and mast cells of psoriasis patients. In the course of pathology of psoriasis, IL-8 not only works as the initiating molecule attracting neutrophils and T cells, promoting the secretion of other cytokines, but also works as the effect molecule stimulating the proliferation of keratinocytes, and promoting the hyperplasia of vessels in dermal papilla. It would be effective to block the function of IL-8 and IL-8 receptor to treat psoriasis. Phage antibody library is constructed by displaying the whole set of antibody genes on phage surface, so it is very convenient to select specific antibodies with different antigens. Furthermore, those phage antibody libraries with large capacity and good diversity have the advantage of obtaining antibodies with high affinity. It has already become one of the key techniques of antibody engineering. Antibodies against IL-8 obtained from phage antibody library would be effective to treat psoriasis.In our present study, prokaryotic expression vector PRSET-IL-8 ofIL-8-His fusion protein was transferred into E coli. BL21 (DE3) using calcium chloride. Single bacterial colonies were picked from the plate and were incubated in LB medium. When it reached the growth period of semilogaithmic, IPTG was added to induce the expression of IL-8-His fusion protein. The bacterial pellet was splited by supersound, the supernatant was passed through a 0.45- m filter and was dialysised with PBS. IL-8-His fusion protein was purified with anti-His antibody affinity chromatography column. After dialysis with PBS, it was analyzed by SDS-PAGE. The result showed that when induced with IPTG on 30 C, IL-8-His fusion protein was expressed in the bacterium periplasm in soluble form, the SDS-PAGE analysis showed the purified protein was present in single strip at the space about 11 000. The density of IL-8-His fusion protein was about 1.5g/L by analysis withspectrophotometer.The purified IL-8-His fusion protein was coated onto immunotube after dilution with coat buffer, phage antibody library was added after blocking with BSA/PBS, XL-Blue in the growth period of semilogaithmic was added after wash with TPBS. After infection about 15min, a small ^amount of bacterial was spread on LB plate containing ampcilin, the left was cultured in an amplified volume. Then under the pressure of ampcilin and kanamycin, the phage antibody was proliferated in the induction of VCSM. In the next day the recovered phages were concentrated as the subsequence phage antibody library and were used in the next screening round. Three rounds of screening were conducted, the tite of subsequence phage antibody library was detected after every screening, the output/input ratio of the phage antibody library was calculated as the index of enrichment of specific antibodies. After the third round of screening, single bacterial colonies were picked from LB plate containing ampcilin and incubated in SB medium, VCSM was added toinduce the expression of phage antibodies, the supernatant was obtained aftercentrifugation. IL-8-His fusion protein was coated onto ELISA board, the expressed phage antibody and HRP-goat anti-M13 antibody was added sequentially, the reaction was showed with OPD substrate. The specificity of positive colonies was tested with IL-8-His fusion protein, human IgG, keratin, BSA and His protein using the same method. The enrichment of the output/input ratio was about 300 times after 3 rounds of screening. After the last round of screening, 60 colonies were picked to produce phag...