Production and Purification of the Endoglucanase Enzyme from Local Isolate Aspergillus fumigatus HBF356

In this study, endoglucanase (EG) from local isolate Aspergillus fumigatus HBF356 was produced and purified using ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography. The molecular weight of the pure EG was determined as 95 kDa. The optimum pH of the purified EG was determined as 4.0 and the optimum temperature as 60°C. It has been observed that the enzyme had a very high thermostability and preserved 75.8% of its activity after 240 hours of incubation at 50 °C. At the same time, the effect of veterinary drugs (gentamicin sulfate and enrofloxacin) on the activity of the EG was investigated. The activity of EG was inhibited by gentamicin sulfate while that was activated with enrofloxacin. The results of this study can give information about the potential of EG from Aspergillus fumigatus HBF356 using as a feed additive and its interaction with animal drugs.

Cloning of Acetoacetyl-CoA reductase and Polyhydroxybutyrate synthase genes from the local isolate Bacillus aryabhattai 6N-NRC into Escherichia coli

Polyhydroxybutyrate (PHB) is the most known degradable biopolymer, produced by some genera of bacteria under unfavorable growth conditions. Isolation and cloning of acetoacetyl-CoA reductase (phbB) and polyhydroxybutyrate synthase (phbC) genes from local isolate previously identified as Bacillus aryabhattai 6N-NRC (GenBank accession no. MH997667.1) was achieved. Suitable primers designed for the phbB and phbC PCR approach were used to clone the phbB and phbC genes. The phbB and phbC genes were successfully isolated, cloned and the PCR amplicon 744 bp and 1089 bp corresponding to phbB and phbC genes were identified, cloned with the pET-29a (+) carrying the phbB and phbC genes, transformed and expressed in Escherichia coli BL21. The amplification of the phbB and phbC genes using specific primers of pET-29a (+) plasmid was performed. The open reading frame of phbB sequence was found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of B. aryabhattai (GenBank accession no. CP024035.1), while the open reading frame of phbC sequence was found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of B. aryabhattai (Gen Bank accession no. CP024035.1) after DNA sequencing. The analysis of the recombinant proteins from E. coli BL21 recombinant colony by tricine-polyacrylamide gel electrophoresis clarified that the expressed phbB and phbC genes in E. coli BL21 strain showed distinct bands of intensity 26.3 KD and 37.5 KD, respectively.

Identification and expression analysis of antigenic sites of hepatitis C virus genotype 3a NS3 and NS5A genes of local isolate

Background Hepatitis C virus, a silent killer, has infected 71 million people globally. The recombinant viral antigenic proteins might be used in the early diagnosis of HCV infection. The NS3 and NS5A genes of HCV function in HCV replication and influence host cellular factors that are involved in HCV pathogenesis. The current study was designed to select NS3 and NS5A antigenic sites, amplified, cloned, and expressed in order to find out better assays for diagnosis or drug and vaccine development. The antigenic sites within NS3 and NS5A genes were selected and confirmed through sequencing and were cloned. The antigenic recombinant proteins were expressed in bacterial strain E. coli BL21ply*, and the expression was confirmed by western blotting by using gene-specific and vector-specific antibodies. Results Specific antigenic regions within the NS3 and NS5A genes of the HCV 3A genotype were amplified. PCR results showed 328 bp and 747 bp antigenic regions, respectively. The regions were confirmed by DNA sequencing and cloned into a bacterial expression vector. Expression analysis showed 12 kDa and 28 kDa of NS3 and NS5A antigenic recombinant proteins, respectively. Taken together, these studies will help to analyze the genetic variability within the local HCV isolates as these antigenic recombinant proteins were quite important in the screening of HCV-infected patients. Conclusions This study might help to enhance the progress in the treatment of HCV infection through the modeling of HCV non-structural genes (NS3 and NS5A) from local isolate, and it might also present the viral genes as potential therapeutic targets.

The effect of heat and disinfectants on the viability of infectious bursal disease virus

Infectious Bursal Disease (IBD) has been reported in Indonesia since 1983 and has become an endemic disease. IBD virus is known to be quite resistant to physical and chemical reagents compared to other viruses, causing this disease is hard to be eradicated. This study aims to evaluate the effect of heat and disinfectants on the viability of the IBD virus. This study was conducted using a local isolate of IBDV obtained from IRCVS. The virus was exposed to heat and disinfectant. Heat treatment was conducted by exposing the virus to 560C and 600C for 30, 60, 120, and 300 minutes. Similarly, the virus was also treated with two disinfectants, virkon and sodium hypochlorite (bleach) for 30, 60, 120, and 300 minutes with different concentrations. Results showed that the virus can be inactivated at a temperature of 800C and 560C for 120 and 300 minutes, respectively. Virkon at a concentration of 1:200 and 1:400 was able to inactivate the virus at 30, 60, 120, and 300 minutes, while sodium hypochlorite 0.5% requires at least 60 minutes to inactivate the virus.

UTILIZATION OF ISOLATED THERAPEUTIC BACTERIA IN TOPICAL OINTMENT FOR INFECTION TREATMENT

This study includes the using therapeutic bacteria isolate in topical ointment for bacterial vaginosis treatment and to compare it with metronidazole ointment that use in a clinical trial under medical supervision. Four active Lactobacillus probiotics bacteria were cultured in %12 skim milk , three of these bacteria were commercial types and one Iraqi local isolate , the total bacterial count of probiotics bacteria were around ≥ 1012 cfu/ml . %10 of individually culture free cells filtrate were added to Muller Hinton broth with secretions of patients infected with bacterial vaginosis "BV", %50 of natural organic components and %50 of local isolate Lactobacillus plantarum cultured in skim milk were mixed to prepare the natural ointment. Eighty-nine BV volunteers were divided into two group, first was 43 volunteers used probiotic ointment, the second group was 46 volunteers used metronidazole ointment "as control group", both groups were under medical observation for two weeks. The results shown that Iraqi local isolate Lb . plantarum gave best inhibition in vitro, according to Amsel criteria the rate of healing with Lb . plantarum ointment group was %97.87, it was higher than metronidazole ointment %91.30 and the healed cases numbers were increased at the day 7 and 14,it has been noticed that the results were positive based on vaginal secretions, and whiff test, clue cell and the pH was above 4.5 shown significant progress. According to Nugent criteria Lb . plantarum ointment gave higher healing percentage which was %95.35.