Home » Autophagy » In order to produce recombinant F1 protein, its coding sequence was amplified which was then cloned in the pTZ57R/T plasmid and followed by verification of the cloning with restriction endonuclease digestion and DNA sequencing

In order to produce recombinant F1 protein, its coding sequence was amplified which was then cloned in the pTZ57R/T plasmid and followed by verification of the cloning with restriction endonuclease digestion and DNA sequencing

In order to produce recombinant F1 protein, its coding sequence was amplified which was then cloned in the pTZ57R/T plasmid and followed by verification of the cloning with restriction endonuclease digestion and DNA sequencing. zoonotic pathogen and its infectious dose is extremely low, estimated between 1 and 10 organisms, which makes it probably one of the most virulent bacteria recognized (2). Clinically, it appears in three different forms including bubonic, septicemic and pneumonic plague (3). Bubonic and pneumonic plague infections are associated with high mortality rate (2,4). If remains untreated, the mortality rate of GSK-2881078 bubonic plague is about 50-90%, and untreated meningitis, pneumonia, or septi-cemia is definitely fatal in most cases.(1). The primary pulmonary plague, although rare, has the mortality rate of 100% if untreated and more than 50% with antimicrobial treatment (5). Consequently, development of efficient, rapid and easy methods for detection of bacterial agent at the earliest time of the infection is necessary (6). In addition, because plague is definitely a fulminating disease and the medical diagnosis is definitely unspecific, the treatment should not be delayed by waiting for bacteriological confirmation or antibody seroconversion which can take more GSK-2881078 than one week (7). generates at 37 C a specific F1 antigen which forms a large gel-like capsule (caf1), readily soluble in the tradition media and the F1- bad phenotype is hardly ever encountered. Previous initial studies possess evidenced F1 antigen in animal cells and serum of one fourth of culture-positive individuals (8). Consequently, various detection methods depend-ing within the Tlr4 detection of F1 antigen or anti-F1 antibodies have been developed to day (9,10,11,12,13). The aim of the present study was to obtain the coding sequence of the F1 antigen and its cloning in a suitable manifestation vector for its manifestation GSK-2881078 and subsequent production of polyclonal and monoclonal antibodies against F1 antigen. MATERIALS AND METHODS Bacterial strains, plasmid and growth condition Cloning process was performed in Top strain (Invitrogen, USA). Proficient cells were prepared by calcium chloride method as described earlier (14,15) and the bacteria were propagated and cultured in Luria-Bertani (LB) (HiMedia, India) medium at 37 C. Whole cell DNA draw out of was from Pasteur Institute of Iran (Tehran, Iran). For T/A cloning, pTZ57R/T plasmid (Fig. 1A; Thermo-scientific, USA) was used. The manifestation vector pBAD/gIII A (Fig. 1B) was purchased from Invitrogen. In order to maintain the stability of the plasmids, ampicillin (Sigma, Germany) was added to the culture medium at a final concentration of 100 g/ml. Open in a separate windows Fig. 1 A; Schematic representation of the pTZ57R/T, B; Schematic representation of the pBAD/gIII A plasmids (from the manufacturers brochure). Primer developing and polymerase chain reaction amplification of the caf1 gene In order to amplify the caf1 coding sequence, specific primers were designed according to the caf1 gene sequence retrieved from Gene lender (Accession quantity: “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_006323.1″,”term_id”:”52788052″,”term_text”:”NC_006323.1″NC_006323.1). Table 1 represents the nucleotide sequences and features of the designed primers. Table 1 Nucleotide sequence and features of the designed primers. Open in a separate windows The primers were prepared when received and utilized for polymerase chain reaction (PCR) amplification of the gene. PCR was performed with Large fidelity PCR enzyme blend (Thermoscientific, USA) and the PCR condition for amplification of the caf1 included a primary denaturation step of 5 min at 94 C, followed by 30 cycles of 1 1 min at 94 C, 45 s at 55 C for annealing, and 45 s at 72 C for elongation. The conditions also included a final elongation step of 10 min at 72 C. PCR products were analyzed using 1% agarose gel elcrophoresis. Cloning and subcloning of the caf1 gene In order to clone the amplified fragment, it was gel purified using GeneJet Gel Extraction kit (Thermoscientific, USA) according to the manufacturer’s training. Then, the purified fragment was ligated to the pTZ57R plasmid using T4 DNA ligase.