title: Effect of compounds on the purification and antibody preparation of the extracellular domain fragment of the receptor CD163.
abstract: BACKGROUND
  Porcine reproductive and respiratory syndrome virus (PRRSV) has been acknowledged as one of the most important agents affecting swine. The scavenger receptor CD163 is one of the important entry mediators for PRRSV.
  
  
  RESULTS
  The tD4 and tD5 CD163 genes were amplified, and the PCR products were cloned into pET-28a(+) (designated pET-28a-tD4 and pET-28a-tD5, respectively). The plasmids pET-28a-tD4 and pET-28a-tD5 were then transformed into the E. coli BL21 (DE3) strain and expressed by adding 1 mmol/L of isopropyl-beta-D-thiogalactopyranoside. The proteins were highly expressed in the supernatant from the tD4- and tD5-producing cells that were incubated with a binding buffer containing the following compounds: β-mercaptoethanol, urea, Tween 20, glycerol, and SDS, while they were rarely expressed in the supernatant from the tD4- and tD5-producing cells that were incubated with binding buffer without the compounds. The tD4 and tD5 proteins were purified, and BALB/c mice were immunized with the purified proteins. Western blotting analysis showed that the tD4 and tD5 proteins were capable of reacting with tD5 antibodies; the titer of both the tD4 and tD5 antiserums was 1:160 against the tD5 protein, as shown by ELISA.
  
  
  CONCLUSIONS
  These studies provide a new way for the purification of proteins expressed in inclusion bodies and the preparation of the corresponding antibodies.
authors: Key Laboratory of Animal Disease Control and Prevention, Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China. guihongzh@scau.edu.cn
authors: Zong-Xi
authors: ZX
authors: Cao
authors: Fu-Rong
authors: FR
authors: Zhao
authors: Kun
authors: K
authors: Jia
authors: Wei-Wei
authors: WW
authors: Sun
authors: Ming-Fei
authors: MF
authors: Yan
authors: Si-Hu
authors: SH
authors: Guo
authors: Pei-Rong
authors: PR
authors: Jiao
authors: Wen-Bao
authors: WB
authors: Qi
authors: Gui-Hong
authors: GH
authors: Zhang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D013552:Swine / Q000235:genetics / Q000276:immunology*; D000098902:CD163 Antigen
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000098902:CD163 Antigen; D011956:Receptors, Cell Surface