Novel ion exchange adsorbents were synthesized by immobilizing sulfopropyl derivative onto homemade highly cross-linked agarose beads. The effects of different ligand densities (from 0.05 to 0.24 mol/L) on static and dynamic adsorption of the adsorbents were investigated using lysozyme as a model protein. Based on these results, rHLF was purified from the transgenic milk by our SP media. 1 mL high density (0.24 mol/L) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF. 5 g/L concentration of rHLF significantly inhibited the growth of Escherichia coli. These studies lay a solid foundation for the wide application of our self-prepared ion exchange adsorbents in protein purification.
Animals ; Cattle ; Female ; Humans ; Lactoferrin ; biosynthesis ; genetics ; Mammary Glands, Animal ; metabolism ; Milk ; chemistry ; Milk, Human ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

Objective: In this study, phage display technology was used to construct the human anti-Zika virus(ZIKV), phage antibody library and to obtain and express the monoclonal antibody. The aim was to master the preparation and expression of human phage antibody library screening method for highly specific antibodies. Methods: The whole blood samples of Zika patients were collected and the lymphocytes were isolated. The RT-PCR method was used to amplify the antibody light chain and heavy chain Fab gene from lymphocyte Ig mRNA. The pComb3H system was used to construct the gene with genetic diversity Preparation of human anti-ZIKV phage antibody library. The purified antibody library was screened by using the purified ZIKV and the obtained ZIKV E protein antigen. Results: The monoclonal antibody Fab fragment gene was successfully obtained for the ZIKV E protein antigen. The gene can be efficiently expressed in Escherichia coli. Conclusions According to the sequence analysis, this study showed that the monoclonal antibody was a new human genetically engineered antibody against ZIKV, which laid the foundation for the early diagnosis of ZIKV, and obtain a specific monoclonal antibody to ZIKV for human treatment of ZIKV infection.