ABSTRACT
Background: Streptokinase A is an antigenic protein secreted by Streptococcus pyogenes. This protein can be used to liquefy pus in pneumonia and the purulent joint swelling and also as an antigen for detection of group A streptococcal infections
Objective: The purpose of this study was expression and production of recombinant streptokinase A of group A streptococci in Escherichia coli in line with diagnostic and therapeutic purposes
Methods: Streptokinase A gene was initially amplified by polymerase chain reaction [PCR] method and sub-cloned into prokaryotic expression vector pET32a. Later, the pET32a vector was transformed into Escherichia coli BL21-DE3-plySs. Gene expression product, induced by IPTG, was purified by Ni-NTA purification kit, and measured by Bradford method. Recombinant SKA was further analyzed by Western Blot. Gene was amplified and sequenced using the Sanger method and the amplified gene in plasmid pTZ57R / T were identical to Recorded sequence in gene bank for streptokinase gene A
Findings: The nucleotide sequence of the gene amplified by PCR was determined by Sanger method. Sequencing results showed similarity in nucleotide sequence of the cloned gene in E. coli with that of group A streptococci available in GeneBank database. The amount of protein product obtained by Bradford method was 3 mg/ml. Recombinant streptokinase protein reacted with mouse sera containing anti-streptokinase A
Conclusion: Our data showed that expression of recombinant SKA protein is possible in Escherichia coli host. The protein product had an approximate molecular weight of 67 kDa with its antigenic properties unchanged. Thus, it can be substituted for ASO and SLO tests used in diagnosis of patients with group A streptococcal infections
ABSTRACT
In molecular diagnosis of microbial agent, purification of chromosome is very important step. In this study, after cell destruction, DNA replication was done by increasing the denaturation time, without DNA purification. In this experimental study eight different dilution of E.coli [8/100, 4/100, 2/100, 1/100, 1/200, 1/400, 1/800 and 1/1600] solution were madce in D.W, Bacteria were separated by filtration. Polymerase chain reaction method was used to propagate 162 rRNA gene by design primers without DNA Purification. In order to confirme sensitivity of PCR, contamination of 15 different sources of Arak well water wafer was compared by MPN method. For confirmed sensitivity of PCR, 15 sources of water in Arak were examined and compared with MPN method. Present of bacteria in diution soution were confirmed by culture. Polymerale Chain reaction [PCR] data were shown this method is able to recognize bacteria in above dilutions after filtration. This study showed high sensitivity of PCR method in compare to MPN method. Results were shown without stages of extraction of DNA, PCR were done without losing chromosome. Therefore false negative results were decrease and avoided from difficult phases