title: [Prokaryotic expression of recombinant bovine IL-4 and development of monoclonal antibodies against bovine IL-4].
abstract: OBJECTIVE
  To express recombinant bovine IL-4 (rBoIL-4) in Escherichia coli and prepare monoclonal antibody (mAb) against rBoIL-4.
  
  
  METHODS
  The IL-4 gene without coding signal peptides was amplified from pSP73-BoIL-4 by PCR, then inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+). The recombinant plasmids pGEX-6p-1-BoIL-4 and pET-BoIL-4 were transformed into DH5α for sequencing. After sequencing confirmation, the two recombinant plasmids were transformed into expression bacteria BL21 and BL21(DE3) respectively. BALB/c mice were immunized with the purified protein rHis-BoIL-4. With the purified rGST-BoIL-4 as detecting antigen, mAb-produced hybridoma cells against BoIL-4 were screened by indirect ELISA. The specificity of the mAbs was characterized by indirect ELlSA, Dot-ELlSA and Western blot.
  
  
  RESULTS
  The recombinant bacteria BL21(pGEX-6p-1-BoIL-4) and BL21(DE3)(pET-BoIL-4) were developed. After induced by IPTG, SDS-PAGE analysis showed that the expression products of rGST-BoIL-4 and rHis-BoIL-4 had a molecular weight of 39 kD and 19 kD respectively, and expressed in inclusion body form. Seven hybridoma cell lines secreting mAbs against BoIL-4, named 2B8, 4A10, 5D6, 5D8, 7G10, 8B7 and 10F8 were obtained. The immunoglobulin subclasses were IgG1. The ascitic titers of these mAbs were 5 000, 16 0000, 10 000, 640 000, 5 000, 40 000 and 40 000, respectively. In Dot-ELISA, all mAbs could only react to the immunogen and the detecting antigen. Western-blot analysis confirmed that mAbs could only react to the corresponding recombinant proteins. The mAbs also reacted to the standard recombinant boIL-4 with biological activity.
  
  
  CONCLUSIONS
  Seven mAbs specific to rBoIL-4 protein are obtained, which may have important application value in further study on diagnosis and pathogenesis of cattle diseases.
authors.affiliation: Key Lab of Zoonosis of Jiangsu Province, Yangzhou University, Yangzhou 225009, China. chenxiang@yzu.edu.cn
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.forename: Cheng-quan
authors.initials: CQ
authors.lastname: Zhang
authors.forename: Zheng-zhong
authors.initials: ZZ
authors.lastname: Xu
authors.forename: Chuang
authors.initials: C
authors.lastname: Meng
authors.forename: Jin-lin
authors.initials: JL
authors.lastname: Huang
authors.forename: Zhi-ming
authors.initials: ZM
authors.lastname: Pan
authors.forename: Xin-an
authors.initials: XA
authors.lastname: Jiao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis*; D000918:Antibody Specificity; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006825:Hybridomas / Q000378:metabolism*; D015847:Interleukin-4 / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D015847:Interleukin-4