bagher yakhchali

The viability and persistence of engineered bacterium candidates in field conditions is one of the consid-erable challenges in the paratransgenesis approach to fighting vector-borne diseases.

In this study two engineered bacterium candidates to produce paratransgenic sand flies, Serratia AS1 and Enterobacter cloacae expressing m-Cherry fluorescent were applied on the leaves of the white saxaul plant (Haloxylon persicum), sugar bait, and rodent burrow soil and their persistent time was tested in desert condition, Matin Abad Coun-ty, Isfahan, August 2022. A PBS suspension of 109 cells/ml was used for sugar bait, spraying on plant leaves (~10 cm2) and 10 cm2 of rodent burrow soil. Sand fly samples were taken daily and were plated on LB Agar and the fluorescent cells were counted after 24 hours.

Time course in general caused a decrease in the number of bacteria for both strains. The two strains were per-sistent in sugar bait and on plant leaves for four days and on soil for two days. Although there were slight differences between the number of the bacteria in sugar baits, which was not significant (P< 0.05). The number of E. cloacae sur-viving on plant and in soil were significantly (P< 0.0001 and P= 0.046) higher than Serratia AS1.

This study shows that plants or sugar bait are useful routes for delivery of the transformed bacteria for the paratransgenesis approach, although, the bacteria ought to be sprayed on plants or sugar baits should be replaced with new ones in four days intervals.

The Forumad chromite area from Sabzevar ophiolite belt, Northeastern Iran, is an environment with high concentration of heavy metals, particularly chromite and magnesite minerals, containing chromium and magnesium.

In this study for the first time, we analyzed and report the diversity of microbial (bacterial and archaeal) community inhabiting in Forumad chromite mine environment using metagenomics approach.

Samples were obtained from different areas of the mine, and total DNA was extracted from water and soil samples. 16S rDNA was amplified using universal primers and the PCR products were cloned in pTz57R/T plasmid. Then, 43% of the positive clones were randomly sequenced. BLAST program in NCBI and EzTaxon databases were used to identify similar 16S rDNA sequences. Phylogenetic analysis was performed using the MEGA5 software and multiple alignments of sequences.

In the phylogenetic analyses, proteobacteria, which contains many heavy metals tolerant bacteria especially chromium, were the dominant population in bacterial libraries with Rheinheimera and Cedecaeas the most abundant genuses. Other phyla were Bacteroidetes, Firmicutes, Verrucomicrobia, Chloroflexi, Actinobacteria, Acidobacteria, Cyanobacteria, Gemmatimonadetes, and Planctomycetes. In the archaeal clone library, all the sequences were related to the phylum Thaumarchaeota. Further, 68.6% of the sequences had less than 98.7℅ similarity with the recorded strains which could represent new taxons.

The results showed that there was a high microbial diversity in the Forumad chromite area. These results can be used for detoxification and bioremediation of regions contaminated with heavy metals, although more studies are needed.

Biotransformation process has been used in various industries due to its ability to produce valuable chemicals and address environmental concerns. Propylene hydroformylation is a process in which n-butyraldehyde and isobutyraldehyde are produced. N-butyraldehyde is a high valuable chemical with many industrial applications, while isobutyraldehyde produced as a by-product is an environmental pollutant. This study offers a biotechnological approach for conversion of isobutyraldehyde into a high-value substance. An engineered strain of Escherichia coli was developed by genomic insertion of alcohol-dehydrogenase gene (adhA) from Lactococcus lactis which can convert isobutyraldehyde into isobutanol. The adhA gene was engineered to substitute some of its amino acids to result in a more efficient enzyme. Engineered gene was synthesized and introduced into E. coli genome to develop recombinant E. coli EG-296 strain. In addition, by using the Qualiteck-4 software, 16 well-defined experiments (L16 Orthogonal array) with two levels of seven variable parameters were used to optimize the process efficiency. The findings of this study revealed that the E. coli strain EG-296 is capable of converting isobutyraldehyde into isobutanol. The optimization results showed that optimum medium composition for the highest isobutanol production were 10 g/L glucose or glycerol as carbon source, 10 g/L NH4CL as nitrogen source, mid-log of inoculum age, and 1% inoculum volume in 25ml medium. After optimization, 560 mg/L isobutanol was produced from 600 mg/L isobutyraldehyde with 91% yield. Recombinant E. coli strain with a relatively optimum medium can be used to remove isobutyraldehyde in refineries or other industries producing this chemical as a by-product.

Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN-β) gene based on the codon preference of the CHO cell in a way to increase the GC content in the third position of each codon.

The nucleotide sequence of the codon-optimized rhIFN-β was synthesized in parallel with the wild-type and expressed transiently in CHO-s cells using Epstein-Bar virus (EBV)-based expression system. The protein expression of the rhIFN-β by codon-optimized and wild-type genes were quantified using ELISA test.

The results indicated a 2.8-fold increase in the expression level of the biologically active form of the rhIFN-β by codon-optimized sequence.

These results shed light on the capability of codon optimization to create a stable CHO cell for scaling up the production of recombinant therapeutics such as rhIFN-β. 

Actinomycetes, Gram positive bacteria, are rich sources of bioactive compounds. Secondary metabolites of these bacteria are used as pharmaceutical compounds Enzymes are one of the most important secondary metabolites which act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly manner. Enzyme production from actinomycetes makes significant facility in industrial and world economic. So, that it is becoming one of the economic indexes of developed countries. The goal of this research is isolation of actinomycetes from Shadegan estuary, optimization of bacterial growth and amylase production. Molecular identification was done by amplification of 16S rRNA gene using R1492 and F27 primers. Optimum growth condition was found at 6% NaCl concentration, 35˚C temperature and pH 8 . Maximum amylase activity in optimum condition was 80150 Unit (one Unit is amount of enzyme that release one microgram of glucose in a minute). The results showed that enzyme production is related to growth of bacteria conditions.

Bactenecin is one of the smallest cationic eukaryotic antimicrobial peptides (AMPs) with a length of 12 residues and a beta-turn structure originated from bovine neutrophil cells. Previous studies reported poor capability of this peptide against Gram-negative bacteria.

The present study aimed at investigating the bactenecin bactericidal activity and determining the effect of increasing the positive charge of amine and carboxyl terminus and increasing the hydrophobicity of the central part of the antimicrobial peptide.

Similar to the native peptide, three designed variants were employed named BM1, BM2, and BM3 that had increased positive charges, hydrophobicity, and a combination of both, respectively. Conditions for purifying and assaying their antibacterial activity against Gram-negative bacteria were predicted and optimized by APD3 and ExPASy servers. The cloned and expressed peptides in Pichia pastoris GS115 were partially purified by anion and cation exchange chromatography. The final purification rate of AMPs by HPLC was reported to be 70% and peptides were further characterized by LC-mass analysis. Finally, a minimum inhibitory concentration test was conducted.

The results implied the more significant effect of positive charges on the performance of these peptides against Escherichia coli.

Gastric cancer is a multifactorial disease. In addition to environmental factors, many genes are involved in this malignancy. One of the genes associated with gastric cancer is CD44 gene and its polymorphisms. CD44 gene plays role in regulating cell survival, growth and mobility. The single nucleotide polymorphism (SNP) rs8193, located in the CD44 gene, has not been studied in gastric cancer patients of the Iranian population. The present study aims to study this polymorphism in 86 gastric cancer patients and 96 healthy individuals. In this cross-sectional case-control study, rs8193 polymorphism was genotyped by allele specific primer polymerase chain reaction (ASP-PCR) technique. The obtained data were statistically analyzed. To find the potential mechanism of action, rs8193 was bioinformatically investigated. rs8193 C allele played a risk factor role for gastric cancer. Patients carrying this allele were more susceptible to have gastric cancer, with lymph node spread. On the other hand, rs8193 T allele, a protective factor, was associated with a higher chance of accumulation in the lower stages of cancer. C allele might impose its effect via destabilizing CD44 and miR-570 interaction. rs8193 is statistically associated with the risk of malignancy, lymph node spread and stage of gastric cancer in Iranian population.

Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. However, the population dynamics of this bacterium with­in An. stephensi and its introduction methods to the mosquitoes have not yet been explored.
Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium.
The E. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. The bacterium was weakly transmitted transstadi­ally from larval to adult stage. The bacterial populations increased smoothly and then dramatically in the guts of An. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time.
Enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. Sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions.

Nowadays, prebiotics are the matter of interest, because of stimulating the growth and activity of beneficial enteric bacteria (probiotic) and their antimicrobial and antitumor characteristics. The aim of this research is to study the effect of prebiotic Chitosan on the growth of probiotic bacteria and their antimicrobial effect.
This research was done in 2010 in order to study of the effect of prebiotic chitosan on the growth of probiotic bacteria Lactobacillus acidophilus PTCC 1643 and Lactobacillus casei PTCC 1608 by using of drawing growth curve of these bacteria in the presence of different chitosan concentrations, also, the effect of this prebiotic on antimicrobial properties of probiotic bacteria was investigated against Escherichia coli by using of overlay method and blank disk method.
According to the test results, the growth of two probiotic bacteria were increased in the presence of chitosan and the most effective concentration of prebiotic chitosan was achieved, 6.5 (mg/mL).Antimicrobial effects of probiotic bacteria were increased in the presence of chitosan against E.coli especially Entrohaemoragic E. coli in comparison with the time that probiotic bacteria were used alone.
According to the results of this study, prebiotic chitosan, due to increasing effect on the growth and antimicrobial characteristic of probiotic lactobacillus, can be a proper candidate for effective symbiotic compound against pathogenic bacteria.

A keratin-degrading bacterium was selected from a bacterial collection of the Najm Biotech Company. Molecular identification indicated that the bacterium is a strain of Bacillus cereus, which can grow and produce keratinase in basal medium containing feather as sole source of carbon and energy. The physicochemical condition (pH, temperature, incubation time, feather concentration) of keratinase production of the isolated B.cereus strain was optimized using response surface methodology (RSM). A maximum keratinase production of 350 U/ml was achieved in 96 h under optimized conditions.

Lipases are versatile biocatalysts with a wide range of application in food, dairy, leather, paper, pharmaceutical and detergent industries. In this study, mesophilic lipase gene from an indigenous Bacillus pumilus F3 that already its gene had been sequenced and identified, was cloned and expressed in methylotrophic yeast Pichia pastoris and codon translation of lipase gene was evaluated in P. pastoris under control of two different promoters (alcohol oxidase (AOX1) and glyceraldehid phosphate dehydrogenase(GAPDH) promoters). In addition, the expression conditions of recombinant lipase F3 was optimized in P. pastoris expression system using BMMY medium at pH=3 ,26ºC in 0.75% methanol. The lipase gene of 648bp with natural signal peptid sequence from B. pumilus F3 and the codon optimized lipase gene cloned and expressed in methylotrophic yeast P. pastoris. The lipase gene was excised from the recombinant plasmid with BamHI, EcoRI enzymes and ligated to the pPIC9 and pGAP9 linearized with the same enzymes. The recombinant plasmids were confirmed by the PCR and restriction enzyme digestion. The Bgl II linearized Ppic9 and pGAP9 recombinant plasmids were introduced into the yeast P. pastoris GS115 genom by electroporation and confirmed by PCR. Lipase expressing yeast was cultivated in a 250-ml shaking flask containing medium expression. Expression of lipase gene was confirmed using p-nitrophenyl palmitate test and SDS-PAGE. Codon optimized lipase produced as well as native gene and lipase expression was low in both. Also, these results suggest that protein structure is more important than codon preference in expression of proteins such as lipases.

Serological methods are commonly used methods for detection of viruses. Preparation of pure viral antigens is a crucial step in production of antibodies required for serological studies. In this research the gene encoding coat protein of a Beet western yellows virus (BWYV) isolate from Iran was amplified by PCR and was ligated into a bacterial expression vector (pET26b) to obtain pET-BWYV-CP clone. Escherichia coli BL21 was transformed with pET-BWYV-CP and expression of the recombinant coat protein was induced by IPTG. The expressed recombinant coat proteins were purified and used as an antigen for rabbit immunization. The antiserum was able to detect recombinant coat protein in total protein extracts of induced E. coli BL21 cells in western blot analysis.

Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function. We introduced a nested-SOE-PCR (N –SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.

Identification and use of more efficient enzymes in the food and pharmaceutical industries is the focus of many researchers. The aim of this study was to search for a new bacterial strain capable of producing high levels of pullulanase applicable to biotechnology, the starch bioprocessing and food industries. A new pullulan hydrolyzing Bacillus strain was isolated and designated SDK2. Morphological and biochemical tests identified the strain as a putative Bacillus cereus strain, which was further characterized and confirmed through 16s rRNA sequencing, and was submitted to GeneBank, under the accession number FR6864500. Quantative analysis of the strain’s pullulanase activity was carried out by the Dintrosalicyclic (DNS) acid-based assay. Thin layer chromatography (TLC) of the culture supernatant, identified the extracellular pullulanase as neopullulanase. Effects of temperature and pH on pullulanase activity were also studied. The optimum conditions for enzyme activity, as represented by 60o C and a pH of 7, resulted in an activity of 13.43 U/ml, which is much higher than some of the previously reported activities. However, growth of B. cereus SDK2 was also observed at a pH range of 5 to 10, and temperatures of 30 oC to 50 oC. The effect of metal ions and reagents, such as Mg+2, Ca+2, Zn+2, Cu+2, Fe+2, Ni+2 on enzyme activity showed that Ca+2 ions increased pullulan activity, whereas the other ions and reagents inhibited pullulanase activity. The ability of B. cereus SDK2 to produce high levels of neopullulanase stable at 60 oC that can generate panose from pullulan, make this newly isolated strain a valuable source of debranching enzyme for biotechnology, the starch bioprocess and medical industries.

In this study، expression of mature Hepatitis C virus core protein (HCVC173)، consistent with a viral isotype isolated in Iran، was studied in E. coli. The 20 kDa- HCVC173 protein was expressed while it contains 18% of total expressed proteins in which a part was partially soluble but the main part was expressed in insoluble inclusion body. However، the produced inclusion body has not shown the expected purity and HCVC173 has been aggregated with the host proteins. Moreover، during refolding of insoluble HCVC173، notable aggregation of the protein was undesirable phenomena. Intramolecular evaluation of target expressed protein predicted two irregularity area in the HCVC173 which may aggregate the expressed protein. Consequently، HCVC173 has the features of the intrinsically unstructured proteins. This character may justify its aggregation during refolding. Moreover، the approach presented in this paper is an alternative solution to overcome the expression limitation of mature form of the HCVC protein.

Microbes particularly bacteria presenting in the gut of haematophagous insects may have an important role in the epidemiology of human infectious disease. The microbial flora of gut and surrounding environmental of a laboratory strain of Phlebotomus papatasi, the main vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in the old world, was investigated. Biochemical reac­tions and 16s rDNA sequencing of the isolated bacteria against 24 sugars and amino acids were used for bacteria species identification. Common mycological media used for fungi identification as well. Most isolates belonged to the Enterobacteriaceae, a large, heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of humans and animals. Enterobacteriaceae groups included Edwardsiella, Enterobacter, Escherichia, Klebsiella, Kluyvera, Leminorella, Pantoea, Proteus, Providencia, Rahnella, Serratia, Shigella, Tatumella, and Yersinia and non Enterobacteriaceae groups included Bacillus, Staphylococcus and Pseu­domonas. The most prevalent isolates were Proteus mirabilis and P. vulgaris. These saprophytic and swarming motile bacteria were isolated from all immature, pupae, and mature fed or unfed male or female sand flies as well as from larval and adult food sources. Five fungi species were also isolated from sand flies, their food sources and coloniza­tion materials where Candida sp. was common in all mentioned sources. Midgut microbiota are increasingly seen as an important factor for modulating vector competence in insect vectors so their possible effects of the mirobiota on the biology of P. papatasi and their roles in the sandfly-Leishmania interaction are discussed.

Citrus tristeza virus (CTV) is among the most destructive pathogens of citrus and causes substantial economic losses in citrus-growing industry worldwide. Considering recent distribution of this pathogen and its capability of transmission by existing aphid vectors in Iran, detection of this virus is enforceable for controlling the damage caused by this pathogen in Iran, as one of the major citrus producing countries. Toward this aim, developing a reliable and sensitive detection method such as enzyme- linked immunosorbant assay (ELISA) would be the first step to detect CTV in large scale screenings of field samples. As the serological method requires great amounts of specific antibody, the consequent preparation of a large scale antigen source for immunization process is necessary. In this study the coat protein gene of CTV (CP25) was amplified by polymerase chain reaction from a cloned CP25 gene in pTZ57R/T and subcloned in pET26b expression vector and named pET-CP25. Two Escherichia coli strains of BL21 and Rosetta Gami (DE3) were transformed by pET-CP25. Expression of recombinant protein was induced by IPTG. The authenticity of recombinant protein was confirmed by western immunoblot analysis using a polyclonal antiserum against CTV particles. The results indicated that CTV coat protein gene was expressed in E.coli. This recombinant protein could be used as a source of antigen for immunization process.

Phytoremediation is a sustainable and environment friendly remediation technique، the potential of which in removal of soil contaminants has been well documented. On the other hand the activity of earthworms helps improve soil conditions. Hence، it is expected that in soil presence of earthworms can enhance the rate of phytoremediation as regards Anthracene. The aim followed in this work was to investigate the effect of a simultaneous application of Eisenia fetida along with Lolium perenne on the remediation of Anthracene pollutant in the soil. The experiments were carried out in the framework of a completely randomized design of seven triplicated treatments. A dose of 500 mg. kg-1 of Anthracene was introduced into the soil. The experiments were continued for a period of 3 months، following which the residual Anthracene was assessed through HPLC. The results revealed that، Eisenia fetida and Lolium prenne could each independently remove 19. 4% vs. 24. 2% of Anthracene، respectively. However، a simultaneous introduction of the earthworm along with an establishment of the plant in soil increased the rate of Anthracene removal up to a complete disappearance.

The species of Pseudomonas genus show different levels of resistance to soil''s heavy metals pollution. Since، there exists is a relationship between heavy metal resistance and plasmid profile of bacteria، in this study، plasmid profile of Plant Growth Promoting Rhizobacteria (PGPR) Pseudomonas fluorescence isolates were studied in the presence of cadmium and zinc. The bacteria were initially isolated from soybean rhizosphere and characterized as based on biochemical and biological properties. Then، different concentrations of Zn and Cd، in the H. E. P. E. S and M. E. S solid mediums، were employed to evaluate the resistance of the isolates to these heavy metals. Based on the growth in the presence of Zn and Cd، the isolates were classified into 4 groups. Two isolates taken from each group were exposed to different concentrations of Zn and Cd for a period of 2 months. Then، the isolates were sub-cultured and their plasmids isolated using alkaline lysis procedure. The plasmid profile of isolates indicated that the toxic effect of the cadmium was higher than that of zinc even when at lower concentrations. At concentrations of cadmium، all the isolates lost their plasmids. Three isolates also showed multiple resistances to heavy metals.

Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l/day. According to the results, the produced mAb had similar affinity to HER2+ tumor cells to that of Herceptin. High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase (DHFR). It is usually accepted that DHFR gene can be amplified in DHFR− CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR.

Urmia Lake, located in the northwest of Iran, is the largest permanent lake in Iran and one of the three permanent hypersaline lakes in the world. Microbial sampling from four western different regions of the lake was done and then, biodiversity of the samples were studied by culture dependent and culture independent methods. In culture dependent methods, halophilic and halotolerant bacteria were isolated under aerobic condition on four growth media, MH, SWN, MHLN and SWNLN. Differentiation of bacterial isolates was performed based on colony features, Gram staining and primary biochemical tests. In culture independent method, environmental DNA from water and soil samples were extracted. After 16S rRNA PCR amplification 16S rRNA library was constructed and 20% of clones were sequenced. Within 217 purified isolates in culture dependent methods, 52 strains were selected for 16S rRNA gene sequencing and representatives of Halobacillus, Halomonas, Planococcus, Gracilibacillus, Bacillus, Pontibacillus, Paracoccus, Marinobacter, Providencia, Staphylococcus, Alkalibacterium, Sanguibacter, Lysobacter, Kocuria, Pontibacter, Salicola, Micrococcus, Oceanobacillus, Brevundimonas, Thalassobacillus, Microbacterium and Piscibacillus genera were identified. In culture independent method, selected clones belonged to Salinibacter، Adhaeribacter and Cesiribacter genera. Discussion and In culture dependent selected strains 18 strains showed less than 98. 7% sequence similarity to the closest known strains and were representatives as new taxa of Urmia lake. In culture independent method selected clones belonged to Bacteroidetes category. Data obtained from this study were similar to other scientific reports from hypersaline lakes.

Lipases (Triacylglycerol acylhydrolases; EC 3.1.1.3) catalyze the hydrolysis of triacylglycerol to glycerol and fatty acids at the interface between water and oil. These enzymes are versatile biocatalysts with a wide range of application in food, dairy, leather, paper, and pharmaceutical and detergent industries. In this study, mesophilic lipase gene from an indigenous Bacillus pumilus F3 was cloned and expressed in methylotrophic yeast Pichia pastoris. The lipase gene of 648bp with natural signal peptid sequence from B. pumilus F3 was amplified by PCR and inserted into the PGEM5Zf vector. The lipase gene was excised from the recombinant plasmid with BamHI and EcoRI enzymes and ligated to the pPIC9 linearized with the same enzymes. The recombinant plasmid was confirmed by the PCR and restriction enzyme digestion. The Bgl II linearized Ppic9 recombinant plasmid was introduced into the yeast P. pastoris GS115 by electroporation and confirmed by PCR. One of the lipase expressing P. pastoris transformants was cultivated in expression medium. The expression of the lipase gene was confirmed by p-nitrophenyl palmitate test and SDS-PAGE.

Infectious Bursal Disease Virus (IBDV) causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger (A. niger). Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG - protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. A number of pyrG+ positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry.

Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful and most of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role of native and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli, the lipase gene (btl2) was cloned downstream of the native Bacillus signal peptide and also in fusion with the pelB, ansB and ansB/asp signal peptides. For this purpose, four recombinant expression vectors (pYRKP.P, pYRKP.N, pYRKP. A and pYRKP.AA) were constructed and expressed in E. coli. Osmotic shock analysis showed that recombinant lipase was overexpressed as inclusion bodies in E. coli. The lipase inclusion bodies were subsequently solublized, refolded and purified using single column ion-exchange chromatography. To evaluate localization of lipase in the cell, the purified lipases were subjected to capillary isoelectric focusing and tandem mass spectrometry. Results showed that all signal peptides were able to direct the lipase from the cytoplasm into the periplasmic space of E. coli, because the periplasmic space of E. coli is not suitable for lipase folding, the translocated lipase aggregates in this space as inclusion bodies.

Human granulocyte macrophage colony stimulating factor (hGM-CSF) has many therapeutic applications. In this study, in order to verify the purification process, the effect of carbon source, IPTG concentration and post-induction time on the secretion of recombinant hGM-CSF into the culture medium by recombinant Escherichia coli during high cell density cultivation were evaluated by using the Taguchi statistical method. The results indicated that glucose, 1mM IPTG and a time of 6 h post-induction, represented optimum conditions. The secreted hGM-CSF, overall volumetric productivity and purified hGM-CSF were 373 mg/l, 18 mg/l/h and 63 mg/l, respectively.