| ?2-microglobulin (?2M) is the light chain of major histocompatibility complex (MHC) class I, which is involved in mutual recognition between cells and induction of immune responses. Due to the patients suffering from chronic renal failure (CRF), the ?2M in their blood cannot be removed by normal metabolism, which is too small to remove it through dialysis, too. So, ?2M becomes a kind of toxin as a result of accumulating in blood, which will lead to dialysis related amyloidosis (DRA). Blood purification therapy is considered to be one of the most effective ways of removing ?2M-However, these existing blood purification materials are of low selectivity, which lead to low efficiency and high side effects. In this paper, we put forward a strategy to create a high selective immunoadsorbent by nanobody from alpaca, and express the nanobody targeting ?2M with high affinity after biopanning. Base on it, the immunoadsorbent is prepared, which has been proved the feasibility when applying to blood purification treatment and removing the redundant ?2M. The research contents of this paper include the following aspects.(1) The biopanning of nanobody anti-?2M-After 4 rounds of biopanning by phage display library technology,233 single phage plaques were selected for ELISA analysis, and only 30 of them showed high affinity. After wiped off repeat and wrong sequences,13 candidates were remained for final ELISA analysis, while 11 of ones were from immune library and 2 from non-immune library.4 nanobody sequences expressed the highest affinity to ?2M, which were A55, A82, A107 and B82.(2) The expression and affinity analysis of nanobodies.4 recombinant plasmids were constructed and transformed into 2 kinds of host cell of E.coli. The expression in Shuffle T7 Express could produce more amount of protein than that of Origami 2. After purifying the protein by Ni-affinity column,6 nanobodies were obtained with more than 90% purity, which were A55S, A107S, B82S, A82S, A55O and A82O. The highest yield was 100 mg/L, belonging to A55S. Data from ELISA and Biacore to qualitative and quantitative analysis identified that A55S had the highest affinity about 2.931 × 10-6 M (KD).(3) The preparation and evaluation of immunoadsorbent. Among 5 kinds of preparation methods, the adsorbent D using ?-polylysine showed the best effect. However, if the ligand density was too high, the nanobody would lose its bioactivity. When loading 2 mg/ml ?2M, the dynamic adsorption capacity of adsorbent D1 was 11.45 mg/mL. However, it would reduce obviously in plasma with the same condition, which was because of the interference from something in plasma.In a word, the nanobody sequences with high affinity are selected from phage display library.8 nanobodies are expressed and the affinities of them are identified. Base on it, the immunoadsorbent is prepared, which has been proved the feasibility when applying to blood purification. However, the non-specific absorption from adsorbent affect the removal of ?2M, and 2 methods are considered of solving the problem, which are improving the affinity of nanobody and site-directed immobilization of nanobody. |
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