The Biopanning And Preparation Of Nanobodies Anti-Leptin

Leptin is a kind of multi-target organ versatile protein hormones. It is involved in energy metabolism, the immune, reproductive and other physiological activities in human body. The abnormalities of leptin level in the blood can lead to obesity or malnutrition, cardiovascular disease, cancer, liver disease and other diseases. Patients with end-stage renal failure are in lower glomerular filtration rate, and the removal of leptin in the body relies mainly on renal metabolism, so most patients with high leptin levels in the blood will have complications of malnutrition, high blood pressure, insulin resistance. Therefore, the development of specific antibodies to achieve leptin detective and removal will have very important clinical significance. Because leptin concentation in the blood is low, we got leptin by prokaryotic expression first, followed by biopanning of phages. The research contains what listed below:(1) The preparation of leptin. Structure recombinant plasmid pET23a-leptin. Then getting the expression conditions of recombinant human leptin protein in E. coli BL21 (DE3):1% of activated cells are transferred to fresh medium, then cultivate cells 8h in shaker at 37℃.Leptin is inclusion bodies in E. coli, which are handled by washing, dissolving, refolding, anion exchange chromatography, finally 95% purified recombinant human leptin is obtained.(2) Nanobody selection. After 5 rounds selection,18 and 5 fragments were isolated respectively from a leptin-immune library and a non-immune library. And one of 5 sequences were included in the 18 sequences. At last we got 22 different sequences of nanobodies. Select 6 kinds of nanobodies with high binding activity after detected by Elisa.(3) The expression of nanobodies. PCR and double digests are used to structure recombinant plasmid pET23a-Nb-anti-LP. Except for Nb-anti-LP-A5, five kinds of Nb-anti-LP were observed soluble expressed in E.coli Shuffle T7 and accounted for 30% of total protein. Then Nb-anti-LP-A5 and Nb-anti-LP-A33 had been purified, High purity (about 90%) of Nb-anti-LP-A5 and Nb-anti-LP-A33 were obtained after immobilized metal-chelated affinity chromatography (IMAC). And both of them showed high binding activity detected by ELISA.