Preparation Of Anti-PTH Nanobody And Its Adsorbent

Parathyroid hormone(PTH) as one of middle molecules is accumulated more than dozens of times in patients with chronic renal failure especially in patients with end-stage renal failure compared to normal people. Too high level PTH results in fracture, muscle pain, anemia and so on, which seriously affects the quality of life in patients. Therefore, it is important to remove excess PTH in clinical. Nanobodies have been widely studied because of its advantages such as low molecular weight, high affinity, high stability, low immunogenicity, and deep epitope. Therefore, this research studied the feasibility of removal of PTH using nanobody adsorbent.1. Selection of expression hosts. W2 and W49 were expressed in E.coli hosts including BL21(DE3), OrigamiTM 2(DE3), Rosseta-gamiTM B(DE3) and Shuffle T7 Express, respectively. According to their soluble expression in different cells, one or two kinds of hosts were selected for following research. At last, the expression of W49 in BL21(DE3) and Shuffle T7 Express were 553.3 mg/L and 272.3 mg/L, respectively. And the expression of W2 in Shuffle T7 Express was 133.2 mg/L.2. Isolation and purification of nanobodies. To obtain nanobodies with a 90% purity level, nanobodies were purified through chromatography technique after the cells were disrupted and centrifugaed. W49 was purified by immobilized metal ion affinity chromatography (IMAC) and its recovery rate in BL21(DE3) and in Shuffle T7 Express was 62% and 58%, respectively. W2 was purified by IMAC combined with strong anion exchange chromatography, and its recovery rate was 46.5%.3. Interaction of nanobodies with PTH. Interaction of W2 and W49 with PTH were studied by ELISA, ITC and SPR. Results suggested that W2 could interact with PTH with an affinity contant about 5?6×10-7 M and W49 could not interact with PTH.4. Adsorbent preparation and its characteristics. W2 was immobilized in Sepharose CL-6B with a density of 9.9 mg/mL gel, which could achieve a maximum binding capacity of 1.8 mg/mL gel and a dynamic binding capacity of 0.98 mg/mL gel for PTH. The elution rate for PTH was 67.3% at the condition of pH 2.0,0.1 M Gly-HCl. The adsorbent was hardly interacted with human serum albumin, while it could interact with a small amount of human immunoglobulin.In summary, immunosorbent with removal of PTH was obtained through this research paper, which provides a theoretical basis for preparation of adsortent with nanobodies as ligand.