Aim: 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.
Conclusion: 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.