Cloning of a serotype-specific antigen from Pasteurella haemolytica A1

Abstract

Recombinant plasmids coding for a soluble (or surface) antigen ofPasteurella haemolytica Al were identified. Two plasmids, both containing the same 5.4 kilobase pairs of insert DNA, were recovered independently by screening a clone bank of P. haemolytica Al genomic DNA in Escherichia coli for the expression of P. haemolytica Al soluble antigens (R. Y. C. Lo and L. A. Cameron, Can. J. Biochem. Cell Biol. 64: [73][74][75][76] 1986). E. coli cells carrying the plasmids were found to be agglutinated by an antiserum raised against the P. haemolytica Al soluble antigens. Analysis of the E. coli clones by electron microscopy revealed patches of amorphous material on the surface of the cells which were not present on the controls. Further characterization with protein A-colloidal gold labeled both these patches and the outer membranes of these cloned cells pretreated with the specific antiserum. These results indicated that the cloned antigen was expressed on the surface of the E. coli cells. The cloned antigen was found to be specific for serotype 1 when tested by slide agglutination against a collection of P. haemolytica typing antisera. Southern blot hybridization, using the cloned DNA as a probe, labeled the genomic DNA from P. haemolytica serotype 1 as well as the cross-agglutinating serotypes 2 and 7, but not DNA from the non-cross-agglutinating serotypes 3 and 4 and PasteureUa multocida. These results demonstrated that serotype specificity could be attributed to the particular antigenic determinants in the genome of the organism.

Genomic Distribution of a Serotype 1‐Specific Antigen‐Coding DNA Fragment of Pasteurella Haemolytica

“…Al-derived insert in plasmid pPHAl (Table 1). These results support the data obtained in an earlier study using P. haemolytica typing antisera obtained from a different source to detect PHAlSAA expression (22). Additional results showed that the serotype 2-derived D N A insert in pPGA2 similarly expressed the PHAlSAA to a high level on the surface of E. coli.…”

“…Isopropylthio-P-D-galactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-!3-D-galactoside (X-gal) were purchased from I'roinega (Madison, WI). Plasmid pPHAl consists of a 3.6 kbp fragment of a PHAlSAA-coding insert subcloned in pBR322 (22).…”

“…Approximately 2,000 recombinant clones comprising the library were screened by colony lift hybridization (22,32). The probe used was a 2.2 kbp HindIII/PstI fragment derived from the pPHAl insert.…”

“…In a previous study, immunologic screening of a genomic library of 5,700 clones led to the isolation of E. coli recombinants expressing an agglutinating, proteinaceous antigen showing serologic specificity to P. haemolyticu A1 antiserum (22). The study also demonstrated surface expression of the antigen by immunoelectron microscopy.…”

An analysis of the codon usage of Pasteurella haemolytica A1

“…DNA fragments coding for P. haemolytica A1 soluble antigens were isolated using an immunological detection procedure as described [24]. Particular recombinant clones expressing the P. haemolytica A1 leukotoxin [19], a serotype-1 specific antigen [22,25] and the glycoprotease [23] have been identified and reported separately. The nucleotide sequences of the cloned P. haemolytica A1 DNA were analyzed using the Pustell Sequence Analysis Software (International Biotechnology Inc.) and the genes lktC, lktA, lktB, lktD, ssaI and gcp have been identified.…”