Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

Background: The homologous recombination [HR] is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.

Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1[+] plasmid.

Results: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing.

Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method.

Cloning and expression of leishmania infantum LPG3 gene by the lizard leishmania expression system

Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum [L.infantum]. The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 [rLPG3] to further research in vaccine designing against leishmaniasis

Il-1 beta [+3953 C/T] and IL-8 [-251 A/T] gene polymorphisms in H.pylori mediated gastric disorders

Previous studies imply that IL-1 and IL-8 gene variations may play a crucial role in the genetic predisposition to different gastric disorders upon H. pylori infection. The aim of this study was to determine the potential association between the prevalence of certain polymorphic sites and the risk of gastric disorders in Iranian population. One hundred and forty three unrelated individuals with different gastric disorders and 374 normal individuals with no gastric disorders and with a negative serology test for H. pylori [control group] were studied for the association between IL-1 beta [+ 3953 C/T] and IL-8 [-251 A/T] gene polymorphisms and H. pylori - mediated gastritis and gastric ulcer. An analysis of genotype frequency for these genes was performed using RFLP- PCR. Based on the data obtained from culture and pathologic findings, the patients were classified into three subpopulations: H pylori [+] non-ulcerative gastritis [+], H. pylori [+] ulcerative gastritis [+] and H. pylori[-] non-ulcerative gastritis [+]. A significantly higher frequency of TT genotype [p=0.02] in IL-1 beta +3953 in H.pylor[+] ulcerative gastritis [+] was revealed compared to the control group. There were no significant differences among other subpopulations. No significant differences in allele and genotype frequencies of IL-8 [-251A/T] were found among the patients. The data suggest that TT genotype in IL- 1 beta +3953 may be a major contributing genetic risk factor for H. pylori induced gastric ulcer. Moreover, the role of other bacterial and host response factors, such as bacterial adherence peptides, host chemokines, and genes involved in gastric acid secretion, must be further investigated in different ethnic populations

Expression of recombinant heat-shock protein 70 of MCAN/IR/96/LON-49, a tool for diagnosis and future vaccine research

Heat shock protein 70 [HSP70] is present in all organisms studied so far, and is a major immunogen in infections caused by pathogens including Leishmania spp. The aim of this study was to clone and express HSP70 from L. infantum strain MCAN/IR/96/LON-49 and evaluate antibody response against HSP70 in visceral leishmaniasis [VL]. The L. infantum HSP70 gene segment was amplified by specific primers. It was cloned into pTZ57R vector and subcloned into pET32a [+] expression vector. The new construct was transformed in the E.coli Rosetta strain, and HSP70 protein was expressed in the presence of 1 mM IPTG and purified using a Hi- Trap chelating column. Antibody responses against HSP70 were determined by ELISA in 37 patients with visceral leishmaniasis and 63 healthy controls. Expression of HSP70 protein was confirmed using SDS-PAGE electrophoresis and dot blot with an anti-His tag antibody. There was no difference between the sequence of nucleotides of the HSP70 gene in the present study and other reported sequences. The ELISA results indicated that the sera of 81.1% [30/37] of the patients and 6.3% [5/63] of controls reacted to L. infantum HSP70. The conservative nature of the HSP70 molecule is an advantage in vaccine studies, because of minor differences [6%] between the nucleotide sequences and consequently the similarity in amino acid sequences in various strains of L. infantum. It could therefore be used in vaccine research against leishmaniasis and also as a tool for serodiagnosis

Interleukin-10 gene polymorphisms and susceptibility to brucellosis in Iranian patients

Interleukin-10 [IL-10] is a Th2-type cytokine that inhibits macrophage activation. It is known that production of IL-10 is affected by its gene promoter poiymorphisms. To investigate the relationship between IL- 10 gene promoter polymorphisms and susceptibility to brucellosis. One hundred and ninety patients with brucellosis and 81 healthy animal husbandmen who owned infected animals and consumed their contaminated dairy products were included in this study. All individuals were genotyped for three bi-allelic IL-b gene promoter polymorphisms at positions -1082[G/A], -819[T/C], and -592[A/C] using polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP]. The distribution of C alleles at positions -592 and -819 of IL-10 were significantly higher in patients than in the healthy animal husbandmen [p = 0.034 and p = 0.0086, respectively]. IL-10 ATA single and double haplotypes were significantly higher in controls, compared to the patients [p = 0.0278 and p = 0.013, respectively]. According to the results higher frequency of C alleles at positions -592 and -819 of IL- 10 in patients may be considered as genetic factors for susceptibility to brucellosis

Polymorphism in the first intron of interferon gamma gene [+874T-A] in Iranian patients with brucellosis

Brucella is a gram-negative bacterium, causing acute and chronic infection in humans and animals. Cell-mediated immunity is the main protective immune response against Brucella spp. Activation of macrophages by IFN-gamma and generation of reactive oxygen intermediates and nitric oxide are the main immunologic mechanisms responsible for control of Brucella infection. To investigate the correlation between IFN-gamma gene polymorphism and brucellosis. 195 patients with brucellosis, 186 healthy patients' family members and 82 healthy farmers who kept infected animals and consumed their contaminated dairy products were selected to take part in the study. IFN-gamma genotyping at position +874 [T to A] was carried out by allele specific polymerase chain reaction [AS-PCR] method. The frequency of AT and TT genotypes significantly increased in farmers compared to patients with rucellosis [P=0.03] while there was no significant difference in genotype distribution between patients and their healthy family members. IFN-gamma [+874] AA genotype is probably a genetic factor that contributes to the susceptibility of the individuals to brucellosis

Modified DNA extraction for rapid PCR detection of methicillin-resistant staphylococci

Nosocomial infection caused by methicillin-resistant staphylococci poses a serious problem in many countries. The aim of this study was to rapidly and reliably detect methicillin-resistant-staphylococci in order to suggest appropriate therapy. The presence or absence of the methicillin-resistance gene in 115 clinical isolates of Staphylococcus aureus and 50 isolates of Coagulase Negative Staphylococci [CNS] was examined by normal PCR. DNA extraction for PCR performance was then modified by omission of achromopeptadiase and proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. All isolates with MIC>8 micri g/ml showed positive PCR. No differences in PCR detection have been observed when normal and modified DNA extractions have been performed. Our modified DNA extraction can quickly detect methicillin-resistant staphylococci by PCR. The advantage of rapid DNA extraction extends to both reduction of time and cost of PCR performance. This modified DNA extraction is suitable for different PCR detection, when staphylococci are the subject of DNA analysis

Distribution patterns of methicillin resistance genes [mecA] in staphylococcus arureus isolated from clinical specimens

There is a growing concern about the application of molecular methods in epidemiological studies of infectious diseases. The objective of this study was to determine the genetic stability of methicillin resistance genes in Staphylococcus aureus for the evaluation of resistance strain distribution. One hundred and fifteen S. aureus isolates from patients with staphylococcal infection were collected. The isolates were screened for methicillin resistance by minimum inhibitory concentration [MIC] examination. The stability of methcillin resistance genes was examined by physical curing and PCR screening methods. The results showed that methicillin resistance Staphylococcus aureus [MRSA] had risen up to 43% in Nemazi Hospital [Shiraz, Iran]. Indeed, the incidence of MRSA in our hospital was 10% during the last four years. The ability to lose [curability] of methicillin resistance genes [mecA] was examined by physical curing method in 49 isolates with MIC >/= 16 micro g ml -1. No sign of curability of mecA gene was observed where 500 colonies from each strain have been studied and exhibited by the same MIC values before and after curing test. Positive PCR results for isolates with MIC >/= 16 micro g ml -1 before and after curing experiment have been achieved. These data confirm the results of curing method, indicating that stable genetic determinants confer methicillin resistance. These results support the hypothesis that resistance isolates may be selected due to clonal selection under antibiotic pressure used in clinics rather than transmission of mobile genetic determinant. The high prevalence of MRSA immerged in our Hospital could be originated due to antibiotic pressure and poor control measures

Simultaneous detection of helicobacter genus and helicobacter pylori species using a multiplex PCR method

In order to improve simultaneous detection and identification of Helicobacter genus in general and Helicobacter pylori specifically and reduce the number of amplifications needed, we established a multiplex PCR. In this study, two pairs of primers: Hcom1 and Hcom2 specific for Helicobacter genus, Hicd1 and Hicd2 specific for Helicobacter pylori species were used. To determine the sensitivity of our multiplex PCR, the lower limits of DNA detection from pure culture were established using phenol-chloroform method. To evaluate the specificity of our protocol, we tested DNA extracted from various Gram-negative and -positive bacteria. A study was subsequently undertaken on stomach tissue samples from 18 patients to evaluate this protocol for detection of Helicobacter in tissue samples. In our optimized PCR, two fragments of 389- and 1200-bp were produced using Hcom1-Hcom2 and Hicd1-Hicd2 primers, respectively. Amplification of Helicobacter pylori genomic DNA was achieved at concentration as low as 0.03 pg equivalent to 150 bacteria. No DNA amplification of other Gram- positive and -negative bacteria was seen. Twelve specimens, positive in culture and rapid urease test for Helicobacter pylori were positive for DNA of this organism using this multiplex PCR. Our results demonstrate that this protocol represents a specific and sensitive assay for simultaneous detection of Helicobacter genus members, in general, and Helicobacter pylori species, specifically, in clinical samples yielding no false-positive