Expression of a Gene Encoding 34.9 kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli

Abstract

Mycobacterium avium subsp. paratuberculosis (Map) contains PPE family antigens which are Proline and glutamic acid rich and may play important role as T cell antigens. Hence the identification and generation of antigens are necessary for immunological characterization. In the present study, the epitopic region of a unique PPE gene encoding 34.9 kDa protein from Map was amplified by polymerase chain reaction. The gene was cloned into Escherichia coli vector pQE30 UA. The recombinant plasmid designated as pQPPE was transformed into E. coli M15 and induced with IPTG revealed the high level expression of 37.1 kDa His-fusion protein (34.9 kDa PPE and 2.2 kDa His-tag), which was confirmed by immunoblotting. Recombinant PPE protein was then purified by Ni-NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant PPE protein reacted with expressed 37.1 kDa His-fusion protein as well as with Map sonicate. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with Map. The results indicated that the recombinant PPE protein of Map was associated with cellular immune response.

Figure 1

Agarose gel 1% of the clone fragment of the gene PPE (1080 bp) in pQE-30 UA expression vector. Lane M1: DNA molecular weight marker 100 bp ladder. Lane1: PCR amplified PPE gene fragment of Mycobacterium avium subsp. paratuberculosis. Lane 2: Released insert of 1080 bp PPE gene fragment after digestion with NheI and EcoRI from recombinant plasmid pQE-30 UA PPE. Lane M1: Presented DNA molecular weight marker Lamda DNA/EcoRI/Hind III digest.

Figure 2

Coomassie Brilliant Blue stained 15% SDS-PAGE showing expressed PPE protein. Lane M: Prestained protein molecular weight marker. Lane 1: Purified recombinant 37.1 kDa his tag recombinant PPE protein. Lane 2: Whole cell extract of E. coli M15 harbouring 37.1 kDa fusion PPE-6xhistag protein (IPTG induced). Lane 3: Whole cell extract of E. coli M15 harbouring 37.1 kDa fusion PPE-6xhistag protein (IPTG uninduced). Lane 4: Whole cell extract of E. coli M15.

Figure 3

Western blot assay of the PPE protein expressed in E. coli cells. Lane M: Prestained protein molecular weight marker. Lane 1: Purified recombinant PPE-protein (37.1 kDa). Lane 2: Whole cell extract of E. coli M15 harbouring 37.1 kDa fusion PPE-6xhistag protein (IPTG induced). Lane 3: Whole cell extract of E. coli M15 harbouring 37.1 kDa fusion PPE-6xhistag protein (IPTG uninduced).

Figure 4

Dot blot assay showing sero reactivity of polyclonal sera against PPE 37.1 kDa recombinant protein with purified recombinant PPE protein and Mycobacterium avium subsp. paratuberculosisi strain 316F sonicate. Lane 1: PBS negative control. Lane 2: Recombinant purified 37.1 kDa PPE. Lane 3: M. a. paratuberculosis strain 316F sonicate.