Preparation Of Site-specific Immobilized Anti ?₂ Microglobulin Nanobody And Its Adsorbent

?₂ microglobulin??₂M?is one of the main toxins of chronic renal failure?CRF?patients.Excessive accumulation of?₂M in patients can lead to dialysis related amyloidosis?DRA?,which seriously affects life quality of patients.Hemodialysis method has limited ability to remove the neutral molecular weight toxins,but?₂M can be better removed by the specific recognition between antigen and antibody.Compared with the traditional antibody,nanobody has the advantages of good stability,high antigen affinity and easy modification,etc.Thus nanobody might be a good biomolecular in blood purification and be used as adsorption material.Proteins immobilized by a traditional non-oriented method often results in a cover-up of the protein active site or even inactivation.Bioenzyme method is a commonly used method of oriented immobilization,which can better maintain the activity of protein compared with non-oriented immobilization.In this paper,aiming at geting an adsorbent for?₂M blood purification,an anti-?₂M nanobody was prepared and oriented immobilized onto agarose by using Sortase A.The contents are as follows:?1?The preparation and activity detection of the anti-?₂M nanobody with a fusion tag of Sortase A identification.Sortase A identified tag and 6×HisTag were added at 3'-terminal of the anti-?₂M nanobody gene sequence by using a genetic engineering method,the modified nanobody was expressed in two kinds of E.coli.By contrast,its expression level in ShuffleT7?DE3?was higher than that in Origami^TM2?DE3?.The nanobody expressed by ShuffleT7?DE3?was purified by immobilized metal ion affinity chromatography?IMAC?,and the yield reached to 60 mg/L.The results of ELISA and Biacore showed that the purified anti-?₂M nanobody has an antigen binding activity with an affinity constant as high as 1.595×10^-8 M.?2?Expression optimization and activity evaluation of Sortase A.A plasmid contained Sortase A gene was transferred into three kinds of E.coli,which can result in soluble expression.Finally BL21?DE3?was chosen as a host bacterium after expression optimization,the expression of Sortase A was 94.1 mg/L with a purity of 95%.The results showed that Sortase A had the catalytic activity by selecting anti-?₂M nanobody with Sortase A identification tag and tripolyglycine labeled by FITC as substrate.By optimizing the enzymatic reaction conditions,it was found that when the molar ratio of nanobody,Sortase A and fluorescence peptide was 5:1:10,the efficiency of enzymatic reaction was up to 60%.?3?Preparation and performance evaluation of blood purification adsorbent.Anti-?₂M nanobody was successfully oriented immobilized on sepharose by Sortase A.Performance between oriented adsorbent and non-oriented adsorbent was compared through the specific adsorption experiment,and the results showed that compared with non-oriented adsorbent,adsorption capacity of oriented adsorbent was increased by 65.6%.The adsorption capacity of unit molecular was 2.4 times than that of non-oriented adsorbent.Oriented adsorbent had good removal rate in the complex system of plasma,but the adsorption performance of adsorbent on removing?₂M was affected by the non-specific adsorption of plasma protein.