title: [Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].
abstract: OBJECTIVE
  To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs).
  
  
  METHODS
  The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells.
  
  
  RESULTS
  The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired.
  
  
  CONCLUSIONS
  Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.
authors.affiliation: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
authors.forename: Ling-Ling
authors.initials: LL
authors.lastname: Yin
authors.affiliation: Blood Disease Institute, Key Labatory of Bone Marrow Stem Cells, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
authors.forename: Su-Hong
authors.initials: SH
authors.lastname: Ruan
authors.affiliation: Blood Disease Institute, Key Labatory of Bone Marrow Stem Cells, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Blood Disease Institute, Key Labatory of Bone Marrow Stem Cells, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.|Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China. E-mail: kainyzhao@163.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhao
authors.affiliation: Blood Disease Institute, Key Labatory of Bone Marrow Stem Cells, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.|Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China. E-mail: limd@163.com.
authors.forename: Kai Lin
authors.initials: KL
authors.lastname: Xu
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics*; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000235:genetics; D053577:Interleukin-1 Receptor Accessory Protein / Q000276:immunology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011946:Receptors, Antigen / Q000235:genetics*; D057127:Single-Chain Antibodies
chemical_list: D000911:Antibodies, Monoclonal; C507765:IL1RAP protein, human; D007135:Immunoglobulin Variable Region; D053577:Interleukin-1 Receptor Accessory Protein; D011946:Receptors, Antigen; D057127:Single-Chain Antibodies