Panning And Identification Of Anti-LC3 Protein Nanobodies

Autophagy is using its own cell vesicles engulfed cytoplasmic proteins or organelles, then contact intracellular lysosomes forming autophagic lysosomal degradation vesicle entrapped contents.LC3 protein, called microtubule-associated protein 1 light chain 3(Microtubule-associated protein 1 light chain 3),is a sign protein of the process of autophagy. LC3 proteins within cells exist in two different molecular weight forms. A molecular weight of 18 KDa are LC3-I,the other having a molecular weight of 16 KDa called LC3- II.When autophagy occurs free in the cytoplasm of I type LC3 after ubiquitin-like changes will be binding(PE) combined to form a type II LC3.In the process of autophagy the LC3-I are constantly reducing and the LC3- II are constantly increase,so it can use the value of the intracellular LC3- II / LC3-I to the evaluation process and extent of autophagy.In this study,recombinant protein LC3 immune healthy alpaca,monitoring serum anti-LC3 antibody titers and collected peripheral blood to isolate its leukocytes and total RNA was extracted white blood cells used in the construction of phage display libraries. Using liquid bead to pan anti-LC3 antibody and expression to identification its activity. The eukaryotic expression vector into the cell to achieve the first time in intracellular use antibody to detect LC3 in autophagy.The main findings are as follows:1. The use of recombinant proteins LC3 immunized alpaca,ELISA results showed that after the third immunization,titers of little change,collected 20 ml alpaca blood after the third immunization isolate its leukocytes and Total RNA was extracted by nested PCR,enzyme digestion and was transformed TG1,obtaining a capacity of 6.8×108 anti-LC3 protein nanobodies gene library. After rescued with helper phage M13K07, the titer of phage displayed VHH library is 5.5 × 1013 CFU/mL.2. Using liquid beads to pan anti-LC3 protein nanobodies, sequencing multiple sequence alignment results showed that the obtained three different nanobody sequence,were named as L338,L340 and L346.3. Construction of pET28a-His-L338, pET28a-His-L340, pET28a-His-L346 prokaryotic expression vector for expression and purification. Using ELISA, Western Blot identification of nanobodies activityand and using Fortebio Octet identification nanobody binding forces of L338?L340 and L346. The results showed that the three anti-LC3 protein Nanobodies were capable of binding recombinant LC3,with affinity of 120 nM,378 nM,and 313 nM.4. Construction of pcDNA3.1-mTagRFP-L338 and pcDNA3.1-EGFP-LC3 eukaryotic expression vector was transfected into Hela cells,adding Rap and CQ respectively induction and inhibition of autophagy,detection of anti-LC3 protein nanobodies in eukaryotic cells activity,the results showed mTagRFP-L338 and EGFP-LC3 can colocalization,proved L338 anti-LC3 protein nanobodies are active in eukaryotic cells,this is first time in intracellular use antibody to detect LC3 in autophagy.