[Preparation and identification of egg yolk antibodies against HLA-A* 0201 heavy chain].

OBJECTIVE: To prepare a high-titer specific and efficient egg yolk immunoglobulin (IgY). METHODS: The antigen of HLA-A* 0201 heavy chain was used to immunize the hens together with Freud's adjuvant and the eggs these hens laid were collected. IgY was purified from the egg yolk by water extraction, salt precipitation with ammonium sulfate and dialysis. The immunoactivity of the IgY was measured by enzyme-linked immunosorbent assay (ELISA) and its purity was determined by SDS-PAGE. RESULTS: The titer of IgY was 1 10(6) as shown by ELISA, with protein concentration of 9.77 mg/ml. The purity of the resultant IgY was more than 90% as suggested by SDS-PAGE analysis. CONCLUSIONS: The antigen of HLA-A* 0201 heavy chain along with complete Freund adjuvant is effective to elicit an immune response of hens for producing IgY antibodies in high yields.

[Preparation and preliminary characterization of soluble HLA-A2-peptide complex].

AIM: To express HLA-A2 heavy chain (HC) and light chain beta(2m) in bacteria and to prepare soluble HLA-A2-peptide complex. METHODS: HC and beta(2m) expressing engineering bacteria was induced for 5 h with 0.5 mmol/L IPTG. After sonification of the bacteria,crude inclusion body was obtained which was then refined, renaturated, ultrafiltrated, and purified by DEAE Sepherose Fast Flow anion exchange chromatography. Then HC and beta(2m) were connected with two specific peptides, purified through Superdex 75 gel filtration, and identified with mAb W6/32 which can recognize native HLA-A2. RESULTS: The expression rates of HC and beta(2m) in engineering bacteria was both about 50%. The purity of both expression products reached to 95%.Moreover, the two HLA-A2-peptide complexes could be recognized by mAb W6/32, even after being stored at 4 degrees Celsius for 2 months. CONCLUSION: The two soluble HLA-A2-peptide complexes prepared by us are stable and lay the foundation for further research of CTL recognition and response.

[Purification and renaturation of recombinant human interleukin-2-pseudomonas exotoxin (IL2-PE66(4GLU)) fusion protein].

OBJECTIVE: To evaluate the effect of a novel approach for purification and renaturation of recombinant human interleukin-2-pseudomonas exotoxin (IL2-PE66(4Glu)) fusion protein. METHODS: A novel purification method established in our laboratory was adopted for the purification of the inclusion body, and after renaturation, recombinant human IL2-PE66(4Glu) fusion protein was purified by DEAE-Sepharose FF ion-exchange chromatography. RESULTS: The purity of the fusion protein that retain its biological activity was as high as 95%, and a recovery rate over 80% of the refolded IL2-PE66(4Glu) fusion protein was achieved. CONCLUSION: The purification and refolding method for inclusion body adopted in this study is simple and practical, which lays the foundation for a large-scale production of the fusion protein.