Search

Female, 8-week-old BALB/c mice were immunized with purified recombinant proteins of the predicted immunodominant region of bovine haptoglobin (pirBoHp). Two monoclonal antibodies (MAbs), named 1B3 and 6D6, were prepared by conventional B lymphocyte hybridoma technique. Titers of ascitic fluid and cell culture supernatant of MAb 1B3 were 1:9.6 × 10(8) and 1:8.2 × 10(4), respectively, and that of MAb 6D6 were 1:4.4 × 10(5) and 1:1.0 × 10(4), respectively. The subtype of MAbs 1B3 and 6D6 was IgG1κ. In Western blot analysis, MAbs 1B3 and 6D6 could recognize the α-chain of native BoHp from plasma of dairy cows. These data indicated that MAbs 1B3 and 6D6 have a potential use for developing diagnostic reagents of BoHp.

The nucleotide sequence of the predicted immunodominant region of bovine haptoglobin (pirBoHp), without the signal peptide sequence, was synthesized based on the codon usage bias of Escherichia coli. The synthesized pirBoHp gene was cloned into the prokaryotic expression vector pET-32a (+), which contains a His-tag. The recombinant pirBoHp protein was successfully expressed in E. coli BL21 (DE3) cells. Western blot analysis showed that the purified recombinant pirBoHp protein could be recognized by an anti-His-tag monoclonal antibody. Further investigations indicated that a polyclonal antibody against the recombinant pirBoHp protein could recognize the α and β chains of native bovine haptoglobin in a pooled plasma sample from dairy cattle suffering from foot rot.

The purified C subunit of the recombinant porcine aminopeptidase N (rpAPN-C) protein was used as an immobilized target to screen potential ligands against rpAPN-C from a 12-mer phage display random peptide library. After five rounds of biopanning, five phage clones showed specific binding affinities to rpAPN-C. In 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays, the phage clone PM1, which contained the HDAISWTHYHPW peptide sequence, had a protective effect against TGEV infection in swine testis cells. Therefore, the HDAISWTHYHPW peptide sequence has a potential use as a small molecular therapeutic agent against TGEV infection.

The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.

The Sel-W gene encoding avian selenoprotein W was cloned into the vector pGEX-6p-1 with GST tag. The recombinant protein Sel-W was expressed in Escherichia coli BL21 (DE3) after induction with 1 mM IPTG. Female 10-week-old BALB/c mice were immunized with the purified Sel-W recombinant protein emulsified in Freund's adjuvant. Two monoclonal antibodies against the Sel-W, 1B8 and 4H5, were produced by lymphocyte hybridoma technique. Subtypes of the MAbs 1B8 and 4H5 were all IgM, and their ascitic fluid titers were 1:2800 and 6400, respectively. In specificity, the MAbs 1B8 and 4H5 showed positive reaction with the recombinant Sel-W protein with GST tag and the synthesized Sel-W protein, and could not react with the GST tag and the recombinant Sel-N protein. In sensitivity, the detection limits of the MAbs 1B8 and 4H5 for the recombinant Sel-W protein and the synthesized Sel-W protein were 39 ng/mL and 52 ng/mL, respectively. These data suggest that the MAb 1B8 and 4H5 will have a potential use for detection and function analysis of the avian Sel-W.