Construction of expression plasmids. All RTBV sequences were derived from the sequenced infectious clone pJIIS2 (32). Plasmids containing the CaMV 35S promoter are derivatives of pTZDH (18). The BamHI site in the polylinker between the CaMV 35S promoter and the CaMV polyadenylation signal was converted into a ClaI site by filling in with Klenow DNA polymerase. Between this ClaI site and the PstI site, we cloned a ClaI-EcoRI fragment covering positions 7403 to 128 (through 8002/0) of the RTBV genome and an XhoI-PstI fragment covering the CAT coding sequence and derived from plasmid pLC15 (17). The EcoRI and XhoI ends of the two fragments were joined with oligodeoxynucleotides, creating the sequences shown in Fig. 1B for pCIC-21 and pCIC-21ATG. To create CIC-12, a suitable oligodeoxynucleotide was inserted between the BstYI and the XhoI sites of plasmid pCIC-21 (Fig. 1B). Deletion of the leader sequence (plasmids diagrammed in panels B and D of Fig. 5 and 6) was accomplished by digestion of pCIC derivates with ClaI and BstBI and subsequent religation. Frameshift mutations (see Fig. 3) were introduced into pCIC-21(ATG) by filling in of either the BstBI site at RTBV genome position 18 (plasmids with the extension Bsfi [Fig. 3]) or the EcoRI site at position 123 (plasmids with the extension Ecfi [Fig. 3]). Mutations around the ORF I start codon (plasmids CIC-12A.M1 to-M6) were introduced by first altering the BstYI site in CIC-12 to a BamHI site (CIC-12A.M1 with the same activity as that of CIC-12A) and then by cloning of suitable oligodeoxynucleotides between the BamHI and the XhoI sites of CIC-12A.M1, creating the sequences shown in Fig. 5. To produce the plasmids diagrammed in panels C and D of Fig. 5 and 6, the XhoI-to-EcoRI (site within the chloramphenicol acetyltransferase [CAT] ORF) fragment of CIC-12A.M1 to-M6 or CIC-21A.ATG was replaced by an XhoI-EcoRI fragment derived from pNRF10Cat (21). This fragment consists of an artificial sequence that adds 14 codons to the 5Ј end of ORF I and is followed by a stop codon, an intercistronic sequence of 70 nucleotides, and the 5Ј end of the CAT ORF with its own ATG start codon, thus creating the ORF organization shown in Fig. 1, 5, and 6.

ABSTRACT. Porcine β-defensin 2 (pBD2) is an antimicrobial peptide in pigs that plays an important role in the immune system by preventing bacterial invasion. To produce an anti-pBD2 antibody, which is not commercially available, we expressed and purified a soluble, his-tagged version of pBD2 (his-pBD2). Purified pBD2 was injected into New Zealand white rabbits to generate polyclonal antiserum. Anti-pBD2 antibodies were purified by ammonium sulfate precipitation, followed by diethylaminoethyl cellulose ionexchange chromatography. The purified polyclonal antibody showed high sensitivity, with a titer as high as 204,800 by enzyme-linked immunosorbent assay, and it also showed high specificity for both his-pBD2 and native pBD2, as assessed by western blotting. Furthermore, immunohistochemistry analysis using the purified antibody revealed that pBD2 protein is distributed in the tongue, liver, kidney, small intestine, and large intestine of pigs. These results indicate that the prepared polyclonal antibody will be a useful tool for further studies of the function and mechanism of pBD2.