RESUMO
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
RESUMO
Hemorrhagic cystitis [HC] in allogeneic bone marrow transplanted [BMT] patients is associated with BK virus [BKV] reactivation manifested as BK viruria. However, since 77-90% of all adult BMT patients excrete BKV, viral reactivation alone cannot be responsible for HC. Recently, a significant overrepresentation of C[right arrow]G mutations in the Sp1 binding site in the non-coding control region [NCCR] of BKV was shown to be present in HC patients and absent in non-HC patients. We aimed to investigate if this mutation resulted in excessive BKV excretion in HC patients. Study design: A Real-Time PCR was developed and used to quantify BKV in urine samples from 21 patients with HC, with and without the mutations, as well as from patients without HC. A Real-Time PCR was developed and used to quantify BKV in urine samples from 21 patients with HC, with and without the mutations, as well as from patients without HC. Quantification of BKV was successful in 18 of 21 urine patients [six with and six without C[right arrow]G mutations] and six patients without HC. A mean of 3.0x10[6] BKV copies/micro was detected in urine samples of HC patients with C[right arrow]G mutations, compared to a mean of 1.5*10[6] BKV copies/micro L in HC patients without C[right arrow]G mutations and a mean of 10x10[6] BKV copies/ micro L in patients without HC. The obtained differences were however not statistically significant, due to one individual non-HC patient with an extremely high BKV copy number. Nevertheless, while 50% of the samples in the HC groups expressed 1*10[6] copies/micro L or more, only one of the samples in the non-HC group contained a virus quantity higher than 5* 10[5] copies. Although we could not confirm that the C[right arrow]G mutations in the Sp1 site of BKV were responsible for an increased viral load in patients with HC, our data suggest that levels of BKV above 10[4] copies/ micro L may indicate a risk for HC