The Expression And Purification Of Human Pth And Preparation Of Immunoadsorbent For The Removal Of PTH

Parathyroid hormone (PTH) is considered as one of the main uremia toxins. High level of PTH can be found in almost all the patients with renal failure and put the patients at a high risk. It badly impacts quality of life and survival rate in the patients. Currently, medication, surgery and blood purification are the three common treatments of high level PTH. Superior to drug treatment and surgery, which place an extra burden on the kidney, blood purification is considered as a promising therapy because of the good effectiveness and fewer side effects may be introduced. However, the existing blood purification therapy exhibits very limited ability in PTH removal. In order to improve the selectivity and safety of the adsorbent, in our study, PTH-specific immunosorbent was prepared with IgY ligand, which is stable, safe and highly specific for human PTH. Additionally, PTH adsorption capacity of the immunosorbent was also investigated. This study consists of following sections:(1) Recombinant plasmids pET23a-PTH and pET21b-PTH were constructed respectively using genetic engineering techniques. PTH was observed soluble expressed in both E.coli BL21(DE3)-pET23a-PTH and E.coli BL21(DE3)-pET21b-PTH. Because of the higher expression level in the former case, pET23a-PTH was selected as the recombinant plasmid for prokaryotic expression. Optimized bacterial culture condition was applied as follows:1%inoculattion and further incubation in LB medium at37℃for3h, followed by induction using0.1mM isopropyl-(3-D-thiogalactopyranoside (IPTG) at37℃for4h. In this condition, the target protein expression level was about0.1mg/mL, up to30%of the total amount.(2) Try to establish a complete separation and purification process suitable for production of PTH. PTH was obtained from E.coli BL21(DE3) through the following steps: ultrasonication,0.12%(w/v) polyethyleneimine (PEI) precipitation, ammonium sulfate precipitation of50%saturation, isoelectric precipitation, SP strong cation exchange chromatography. Analysis of the final product by SDS-PAGE, SEC-HPLC and RP-HPLC suggests a high purity above95%. Meanwhile, the molecule weight of the final product was detected to be9406.777Da by MALDI-TOF-MS. Immunofluorescence analysis demonstrated that the purified PTH has good immunogenicity with anti-PTH antibody.(3) Laying chickens were immunized using purified PTH as the antigen. As a consequence, production of specific IgY in egg yolk was confirmed by Western blot and indirect ELISA analysis. After preliminary extraction from egg yolk, the IgY antibodies were further purified to92%by an affinity column, which was prefunctionalized with PTH ligands. The purified antibodies were linked to CDI-Sepharose to prepare immunoaffinity adsorbent. According to our results, PTH in the serum samples of patients with hyperparathyroidism can be effectively removed by the immunoaffinity adsorbent, as a efficiency of40-60%was observed.In this study, the specific IgY was employed as ligand to prepare immunoadsorbent. The immunoadsorbent was confirmed to be effective in PTH removal from the blood of different individuals, therefore shows great potential for the treatment of hyperparathyroidism.