Molecular cloning of a developmentally regulated protein isolated from excretory-secretory products of larval Dirofilaria immitis.

Three proteins isolated from the excretory-secretory products (ES) of larval Dirofilaria immitis have been previously characterized and termed the 20, 22L and 22U kDa proteins. Two of the proteins (20 and 22L) were produced and released around the time of the third molt and were specifically recognized by immune dog sera. An amino acid sequence common to both proteins was used to synthesize a DNA probe to molecularly clone these molecules from a 48-h third stage larval cDNA library. The DNA sequence of the isolated clones encoded a 17.5 kDa protein with a 21 amino acid hydrophobic leader sequence that when removed yielded a 15.3 kDa protein starting with the N-terminal sequence obtained from the 20 kDa protein and containing all sequences obtained from tryptic peptides of the 20 and 22L kDa proteins. It was hypothesized that the 20 and 22L kDa proteins were the same, differing only by a 21 amino acid hydrophobic leader sequence which was later cleaved. The calculated molecular masses were consistent with those determined by reducing Tris-tricine SDS-PAGE. Expression of the protein without the leader sequence was accomplished in Escherichia coli. Antiserum raised against the expressed protein demonstrated the presence of the protein in L3 and L4, but not in adults or microfilariae. Expression of the protein with the leader sequence using a baculovirus system demonstrated processing of the signal sequence at the same time as found in larval D. immitis ES. Sera from dogs immune to infection were reactive with the D. immitis proteins expressed in either E. coli or insect cells.