Introduction

Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human (whooping cough). Bordetella pertussis has some important antigens such as pertussis toxin, adenylate cyclase, fimbriae, agglutinogens, Filamentous Hemagglutinin (FHA), pertactin and Outer Membrane Proteins (OMP). Pertussis toxin (PT) is also called lymphocytosis- promoting factor, histamine-sensitizing factor, islet activating protein, and pertussigen (1-3).
Although pertussis is a vaccine preventable disease, according to the World Health Organization (WHO), there are an estimated 50 million cases of pertussis per year worldwide, with approximately 300,000 leading to death (4).
There are two different types of pertussis vaccine, whole cell and acellular vaccine (1,4). Vaccination with whole cell of Bordetella pertussis causes some side effects e.g. a serious allergic reaction fever, prolonged seizures, decreased consciousness, lasting brain disease, or death. Thus, many reports have been focused on acellular vaccine as a potent effective substitute, lacking the side effects. Pertussis toxin is thought to play a major role in the pathogenesis of whooping cough. It is also believed to be a major protective antigen, so is one of the major acellular pertussis vaccines (5).
PT can be divided into two different A and B parts. Part A is the enzymatically active S1subunit while B is composed of S2, S3, (S4)2, and S5 subunits. The B part binds to the receptor of eukaryotic cells and allows the entry of the enzymatically active S1subunit into the cells (1,6,7). After internalization, S1 transfers the ADP-ribose moiety of NAD to the Gi regulatory component of the adenylate cyclase complex. The Gi ADP-ribosylation decreases the response to inhibitory agents of adenylate cyclase and increases the response of stimulatory agents (1,8-11).
In this study, as S1 is the most important immunogenic part of PT, it was cloned in suitable vectors and the recombinant S1 (rS1) was expressed in E.coli as a prokaryotic host cell. Finally, expressed rS1 was confirmed by monoclonal and polyclonal antibody against pertussis toxin.

Materials and Methods

Bordetella pertussis strain 18323 (ATCC 9797), a good producing PT and highly pathogenic strain (MAST, Germany), E.coli DH5α as cloning host, BL21 (DE3) (Novagen, USA) as expression host, pET-22b(+), pET-14b (Novagen, USA) and pAED4 as expression vectors, SmartTaq DNA polymerase ,T4 DNA ligase, dNTPs (Cinagen, Iran), Pfu DNA polymerase, NdeI and XhoІ (Fermentas, Lithounia), monoclonal antibody against pertussis toxin (Abcam, USA), polyclonal antibody against pertussis toxin (NIBSC, England) and HRP conjugated antibody against mouse IgG (Sigma, USA) were used in this study.

Bordetella pertussis culture and genomic DNA extraction
Bordetella pertussis was cultured in Bordet-Gengou agar for 3 days at 37°C. The bacteria were harvested in PBS and then were heated at 56°C for 30 min .Genomic DNA was extracted from pellets by DNA extraction kit (high pure template DNA extraction kit, Roche Applied Science, Germany) according to the manufacturer's instruction.

Primer designing and amplification of S1 gene
PT DNA sequences of Bordetella pertussis from Genbank was retrieved and aligned by DNAMAN software (Version 4.13). On the basis of Bordetella pertussis 18323 DNA sequences (GenBank: AJ506996.1), two specific primers were designed with Oligo software (version 5) for amplification and isolation of the S1 full length. The upper and the lower primers were flanked by NdeI and XhoI restriction sites, respectively. These enzymes allow cloning of the gene in pET system vectors. The sequences of the primers were:
NdeI_Bor_S1 (Forward):
5- GAATTCCATATGCGTTGCACTCGGGC-3
XhoI_Bor_S1 (Reverse):
5-CCGCTCGAGGAACGAATACGCGATGCTTT-3
PCR was carried out in Mastercycler gradient (Eppendorf, Germany) using Pfu DNA polymerase. The PCR reactions were carried out in 50 µl containing: 1 µl purified Bordetella pertussis genomic DNA, 5 µl 10X PCR buffer (100 mM Tris-HCl, 15 mM MgCl2 and 500 mM KCl), 200 µM dNTP, 0.2 µM of both primers and 2 U of Pfu DNA polymerase. The initial denaturation step was at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, 1 min annealing at 64°C and 1 min elongation at 72°C. The final elongation step was performed at 72°C for 10 min.

Cloning of amplified S1 gene
Amplified PCR product was purified by high pure PCR product purification kit (Roche Diagnostic, Germany). Both of S1 fragment and pET-22b(+) were double digested with NdeI and XhoI. After cleaning with PCR product purification kit (Roche Diagnostic, Germany), they were ligated to each other by T4 DNA ligase at 22°C. DH5α was transformed with the ligation mixture via heat shock. The positive clones were screened on ampicillin/LB agar. The recombinant S1-pET-22b(+) plasmid was purified by high pure plasmid purification kit (Roche Diagnostic, Germany).
Ligation mixtures were transformed to E.coli DH5α. All of insertion steps of S1 gene are done individually for all of the mentioned vectors. The S1 gene that had been inserted in purified plasmid pET-22b(+) was sequenced by MWG Company (Germany). The sequence was compared with Bordetella pertussis 18323 S1 gene sequences (GenBank: AJ506996.1) for homology by BLAST analysis. The vectors were transformed in BL21 (DE3) as expression host by heat shock method.

Expression of recombinant S1 (rS1)
Expression of recombinant S1 was in LB broth and optimized by addition or adjusting of some component such as glycine, glycerol in LB broth, use of different incubation temperatures (4°C, 25°C, 30°C, 37°C) or different concentrations of IPTG (0.1 to 1 mM) as inducer.
In brief, 50 ml of media were inoculated by 1.5 ml of an overnight fresh culture of expression host. Induction was carried out when culture had reached the OD of 1 at 600 nm. Zero, 3 and 6 hrs and overnight samples immediately were centrifuged at 4°C at 12000 rpm for 5 min. Pellets were resuspended in 2x SDS-PAGE sample buffer and stored at -50°C. Expression of different mentioned vectors were studied in E.coli BL21 (DE3) as expression host. All of samples (pellets and supernatants) were run by SDS-PAGE electrophoresis following by Coomassie Brilliant Blue for pellets and silver nitrate for supernatants.
Western blot assay
The SDS-PAGE pellet of BL21 (DE3) with rS1-pET-22b(+) were wet transferred on nitrocellulose for 1 hr at 100 volt. Monoclonal and polyclonal antibodies against pertussis toxin were used as the primary antibody and rabbit HRP conjugated IgG against mouse IgG as secondary antibody. 4-chloro-1-naphthol was used as substrate for visualization of bands.

Result

Genomic DNA isolation and PCR amplification of S1 gene
Genomic DNA from 72 hrs grown Bordetella pertussis cells was extracted successfully. The quality and purity of the extracted DNA in Nanodrop and also on agarose gel electrophoresis were good. S1 gene was isolated by PCR amplification using the designed primers. The optimized PCR amplification isolated an intense single 810 bp band (Figure 1).
The fragment contained NdeI and XhoI overhangs on the 3' and 5' of the gene, respectively to ligate it in accurate direction for expression.

Cloning and sequencing of S1 gene
Both amplified fragment and pET-22b(+) were purified and digested with NdeI and XhoI for ligation to each other. The ligated plasmids were selected on the basis of ampicillin resistance. Plasmids which were extracted from positive clones showed two fragments, one about 810 bp (inserted S1 gene) and the other one about 5400 bp (plasmid without inserted S1 gene) in digestion with NdeІ and XhoІ (Figure 1).
Restriction analysis of the isolated fragment with NcoІ, an extra confirmation test for the isolated S1 gene, produced two expected fragments; a 730 bp and 80 bp and confirmed the accuracy of S1 sequence (data not shown). This restriction analysis was used to control and trace the gene in all steps of purification, digestion and ligation.
Constructed vectors were extracted, purified and sequenced bidirectional to have the highest possible accuracy. Nucleotide sequencing of inserted gene had 100% homology with nucleotide sequence of Bordetella pertussis 18323 S1 gene (GenBank: AJ506996.1).

Expression of recombinant S1 (rS1)
S1-pET-22b(+) construct was able to express S1 in relatively high level, enough to be observed obviously on SDS_PAGE with Coomassie Brilliant Blue staining (Figure 2). Cell extract and supernatant were studied to trace any possible expression. Two distinct bands did exist in cell extract with 28 and 31 kDa estimated bands. No secretory expressed protein was observed in the gel electrophoresis of supernatant. In order to indicate any possible expression, the supernatant SDS-PAGE gel was re-stained by silver nitrate because of its higher sensitivity. No obvious extra cellular expression was observed (data not shown). By optimization of culture conditions, the best condition for expression rS1-pET-22b(+) in E.coli BL21 (DE3) were:
LB broth as media, 30°C incubation temperature, 250 rpm rotation, 6th hr culturing and 0.2 mM IPTG as inducer. In these conditions two distinct and separate molecular weight proteins (about 28 and 31 kDa) were detected (Figure 2).
S1-pET-14b and S1-pAED4 constructs were not able to express S1 in enough level (Figure 2).

Western blot assay
Both of the different bands (about 28 and 31 kDa) were confirmed as S1 in western blot analysis (Figure 3). This test was repeated with polyclonal primary antibody against PT instead of monoclonal primary antibody against PT and both mentioned bands were detected (data not shown).

Discussion

Pertussis vaccine has a crucial role in the control of highly contagious whooping cough disease. It has enough importance to attract many scientists. Thus, many studies have been done on bacteriology, physiology, immunology and pathology of this bacterium. Currently, two types of vaccines including whole cell and acellular vaccines are being used worldwide. Developed and some of the developing countries are switching to use acellular one; because of the side effects of whole cell vaccines. S1 subunit, one of the most important immunogens of pertussis toxin (an essential components of acellular pertussis vaccine), can be used as a good candidate for this purpose. Some studies attempt to express the recombinant S1 in different hosts such as E.coli (2,9,12,13). However, because of the toxic nature of this protein its expression is relatively a tedious work.

Conclusion

In future, we are going to evaluate the immunogenicity of these fragments (in vivo and in vitro) in order to investigate the possible protectivity as candidate immunogens for acellular pertussis vaccine. In a parallel study the antigenicity of them as a specific marker of anti pertussis antibodies could be investigated for detection of any current or previous pertussis infection.

Acknowledgement

This research was supported by a grant from Razi Vaccine & Serum Research Institute, (Karaj, Iran) and Tarbiat Modares University (Tehran, Iran). We appreciate Mariagrazia Pizza and Silvana Savino for their helpful gift (monoclonal antibody against pertussis toxin) from the Research Center of Novartis Vaccines in Italy.