Preparation And Characterization Of Monoclonal Antibody Against Mature Prion Protein Of Qinchuan Yellow Cattle

Objective: To obtain monoclonal antibody (mAb) against mature prion proteins(PrP)of Qinchuan yellow cattle, the purified and refolded recombinant bovine mature prion proteins were regarded as immunogen, so that it will establish base for BSE immune diagnosis reagent kit.Method: Transforming recombinant plasmids(pET30a-PrP(25~242aa) contained Qinchuan yellow cattle PRNP and conserved in the lab into E. coli JM109(DE3), the expressed products were obtained from reombinant bacterium induced by IPTG and were identified correctly by SDS-PAGE analysis. Since then, the fusion proteins were purified by Ni-NTA immunoaffinity chromatograghy. The recombinant expression products were renatured by dialysis and their protein concentration were mensurated. As import mAb SAF70 for detect antibody, Western blot analysed reactiongenicity and proteinase K sensitivity of the fusion proteins. The purified and renatured fusion proteins of bovine mature PrP as immunogene (positive immuogene) and proteins of E. Coli JM09 (DE3) containning pET30a vector as comparison immuogene (negative immuogene) were emulsified with Freunds complete adjuvant respectively. 8-week-old clean class Balb/c female mice were immunized by hypodermic injection, 100μg per mouse. On the fourth and eighth week, the coequal dosage immunogenes emulsified with Freunds incomplete adjuvant were hypodermic injection. On the twelfth week, the immunogenes diluted by PBS were celiac injection, 150μg per mouse. After three days, the spleen cells of immunized mice were fused with myeloma cell line SP2/0 under PEG1500 and incubated in HAT culture liquid. After 10~15d, the cell liquids were determined by ELISA. ELISA plates were coated by the purified fusion proteins of bovine mature PrP and proteins of E. Coli JM09 (DE3) containning pET30a vector and the coated concentration was 25μg/mL.The antiserum(1:960) of mice immuned with positive immuogene and import mAb SAF70 (1:300) were positive comparison. The antiserum (1:960) of mice immuned with negative immuogene and SP2/0 cell liquids (1:1) were negative comparison. Goat anti Mouse IgG-HRP was the second antibody. The cells was thought of as positive clone as long as OD490nm value of the hybridoma cell liquids/negative comparison preponderated over three times. N64 hybridoma cell was limit diluted. After three subclone, N64 hybridoma cell was preserved in liquified nitrogen gas and cultivated continuously for three months to determined the stability of cell excretive antibody. To prepare ascites antibody, 1×10~6 N64 hybridoma cells were injected into pretreatmnt BALB/c mice by germfree liquid olefins. Adopting Protein A Agarose affinity chromatograghy, the ascites antibody could be purified. Using double-antibody layer ELISA, indirect ELISA and Western blot, Ig type and subtype, titer, relative affinity and specificity of the ascites antibody were determined and analyzed respectively. Finally, the epitope of the target proteins recognized by mAb was roughly identified through epitope mapping.Result: Monoclonal antibody (mAb) against PrPC mature protein of Qinchuan yellow cattle is obtained. The mAb belongs to IgG2a ,its titer is 1×10~5 and its relative affinity is 0.2μg/mL. Western blot demonstrates that the mAb has stronger specificity . Epitope analysis shows the mAb only reacts with carboxyl-terminal of recombinant PrP.