Preparation Of Site-specific Biotinylated Bivalent Anti-PTH Nanobody And Its Adsorbent

Parathyroid hormone(PTH)is one of the molecular toxins accumulated in the body at the end of chronic kidney disease.Excessive PTH lead to secondary hyperparathyroidism,causing multi-system damage to the patient.Currently,PTH can not be removed by hemodialysis,thus specifically PTH blood purification adsorbent is needed.Nanobody,which has the advantages of easy transformation,high affinity and excellent blood compatibility,etc.,is the ideal material for preparation of blood purification adsorbent.In addition,biological properties of nanobody can be significantly improved through a series of modification.And streptavidin biotin system is a site directed immobilization method commonly used in protein immobilization,which can conducive to maintain protein activity.In this paper,PTH blood purification adsorbent was prepared by using site-specific biotinylated bivalent anti-PTH nanobody.And the feasibility of its application to PTH blood purification was explored.The contents include the following aspects.(1)Preparation of bivalent anti-PTH nanobodies.The avi-tag bivalent anti-PTH nanobody Nb1-Nb2 and monovalent anti-PTH nanobody Nb1 were constructed by selecting the excel ent sequence of five anti-PTH nanobodies in our laboratory.The avi-tag modified nanobody was expressed in three kinds of E.coli.And the soluble protein produced by E.coli Shuffle T7 was purified by immobilized metal ion affinity chromatography(IMAC).The final yield of the two nanobodies could reach about 100 mg/L with purity of more than 85%.Biacore was used to determine the affinity of two nanobodies.It was found that the affinity of Nb1-Nb2 was 6.2 times higher than Nb1,and the KD reached 3.757 × 10-7 M.The avi-tag nanobody Nb1-Nb2,Nb1 was biotinylated in vitro under the action of biotin ligase,and the catalytic efficiency is up to 80%.(2)Preparation of streptavidin.Streptavidin plasmid was transferred into E.coli Shuffle T7,the expression of soluble proteins were purified by IMAC,and streptavidin purity is as high as 95%.The activity of streptavidin is 12.5 U/mg with high biotin binding activity,which was determined by 233 nm UV spectroscopy.(3)Preparation and evaluation of adsorption performance of the PTH adsorbent.The PTH blood purification adsorbent was prepared through oriented and non-oriented conjugation of biotinylated anti-PTH nanobodies on streptavidin modified agarose gel.The adsorption isotherms of the adsorbents were measured by two methods,and the maximum static adsorption capacity was 2.29 mg/mL.The adsorbent properties of the adsorbents were compared by dynamic adsorption method.As a result,the adsorbent capacity of the adsorbent prepared by oriented immobilization of Nb1-Nb2 can reach to 0.565 mg/mL,which is much higher than that of the non-oriented immobilized adsorbent.However,in the complex environment of plasma,there are more non-specific adsorption,the adsorption capacity of PTH were decreased.