• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.561-566
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• 2014

The complete coding sequence of Wild Argali ISG15 cDNA was generated by rapid amplification of cDNA ends. The ISG15 cDNA was 642 bp with an open reading frame of 474 bp, which encoded a 17.47 kDa protein composed of 157 amino acids. Its amino acid sequence shared 97.9%, 80.8%, 91.4%, 94.3%, 78.3% identity with those of ISG15cDNA from Ovis aries (accession no. NM001009735.1), Capra hircus (accession no. HQ329186.1), Bos taurus (accession no. BC102318.1), Bubalus bubalis (accession no. HM543269.1), and Sus scrofa (accession no. EU647216.1), respectively. The entire coding sequence was inserted into the pET-28a vector and expressed in E. coli. The recombinant protein corresponded to the expected molecular mass of 25 kDa as judged by SDS-PAGE, and it was detected in the bacterial inclusion bodies. The expressed protein could be purified by $Ni^{2+}$ chelate affinity chromatography and the results from the lymphocyte proliferation test showed that the product could stimulate lymphocyte proliferation very well (p<0.05), which further confirmed its biological activity.

• Parasites, Hosts and Diseases
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• v.60 no.3
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• pp.201-205
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• 2022

Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome c oxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/µl B. microti genomic DNA. No cross-reaction with any other blood-transmitted parasites was observed. Our results demonstrated that the fRAA assay would be rapid, sensitive, and specific for the detection of B. microti.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1620-1627
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• 2009

The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation-exchange chromatography under denaturing conditions, followed by refolding together with size-exclusion chromatography and gradual dialysis. The resulting yield of the soluble GSTAHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.71-76
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• 2009

Salivary lipocalin (SAL1) is a member of the lipocalin protein family that has a property to associate with many lipophilic molecules. The importance of SAL1 during pregnancy in pigs has been suggested by our previous study which has shown that SAL1 is expressed in the uterine endometrium in a cell type- and implantation stage-specific manner and secreted into the uterine lumen. However, function of SAL1 in the uterus during pregnancy in pigs is not known. To understand SAL1 function in the uterus during pregnancy, we generated recombinant porcine SAL1 protein in an insect cell line. Porcine SAL1 cDNA was cloned into a baculovirus expression vector using RT-PCR and total RNA from uterine endometrium on day 12 of pregnancy, and the expression vector was used to generate recombinant Bacmid containing the SAL1 gene. The recombinant Bacmid was then transfected Sf9 cell to produce recombinant baculovirus. By infecting Sf9 cell with recombinant baculovirus, we established a SAL1-expressing insect cell expression system. Immunoblot analysis confirmed SAL1 expression in the infected cells. Recombinant SAL1 produced by the Sf9 cell line will be useful for understanding physiological function of SAL1 during pregnancy in pigs.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.989-992
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• 2003

Recombinant human tissue plasminogen activator deletion mutein (Reteplase) is a clinically promising thrombolytic drug. Reteplase cDNA was subcloned into a bacteria expression system, and the resultant recombinant was biologically characterized. The Reteplase was expressed in Escherichia coli as an inclusion body, and the downstream processes of the Reteplase inclusion body included denaturation, renaturation, and purification. A protein disulfide isomerase (PDI) was used to assist the refolding of Reteplase, and it was found to increase the refolding rate from less than 2％ to more than 20％. The refolded Reteplase was purified through two chromatography steps, including lysine-coupled agarose affinity chromatography and then CM-sepharose cation-exchange chomatography. The purity of r-PA was analyzed by Western bolt analysis, and N-terminal amino acid and amino acid composition analyses confirmed the end-product. Reteplase showed higher thrombolytic potency in an animal thrombus model.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.1
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• pp.77-82
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• 2014

The study aimed to evaluate the potential effects of feeding with phytase transgenic corn (PTC) on organ weight, serum biochemical parameters and nutrient digestibility, and to determine the fate of the transgenic DNA in laying hens. A total of 144 50-week-old laying hens were grouped randomly into 2 treatments, with 8 replicates per treatment and 9 hens per replicate. Each treatment group of hens was fed with diets containing 62.4% non-transgenic conventional corn (CC) or PTC for 16 weeks. The phytase activity for CC was 37 FTU/kg of DM, whereas the phytase activity for PTC was 8,980 FTU/kg of DM. We observed that feeding PTC to laying hens had no adverse effect on organ weight or serum biochemical parameters (p>0.05). A fragment of a poultry-specific ovalbumin gene (ov) was amplified from all tissues of hens showing that the DNA preparations were amenable to PCR amplification. Neither the corn-specific invertase gene (ivr) nor the transgenic phyA2 gene was detected in the breast muscle, leg muscle, ovary, oviduct and eggs. The digestibility data revealed no significant differences between the hens that received the CC- and PTC-based diets in the digestibility of DM, energy, nitrogen and calcium (p>0.05). Phosphorus digestibility of hens fed the PTC-based diet was greater than that of hens fed the CC-based diet (58.03% vs 47.42%, p<0.01). Based on these results, it was concluded that the PTC had no deleterious effects on the organ weight or serum biochemical parameters of the laying hens. No recombinant phyA2 gene was detected in muscle tissues and reproductive organs of laying hens. The novel plant phytase was efficacious in improving the phosphorus digestibility of laying hens.

• Journal of fish pathology
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• v.22 no.2
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• pp.147-153
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• 2009

To study the biological activity of interleukin-$1\beta$(IL-$1\beta$), a proinflammatory cytokine, in nile tilapia, Oreochromis niliticus, the recombinant tilapia IL-$1\beta$ was produced in E. coli cells based on pQE vector. Ni-NTA (nitriloacetic acid) metal affinity chromatography was used to purify recombinant protein. The eluted fractions exhibited a single band of protein with a molecular weight of about 25kDa, which is in close agreement with 25.4 kDa predicted by the cDNA sequence. The biological activity of the purified recombinant tilapia IL-$1\beta$ was tested through its effects on IL-$1\beta$ gene expression, which are known as IL-$1\beta$ inducible genes in mammals and fishes. IL-$1\beta$ gene expression induced by poly I:C, a synthetic double stranded RNA, was also assessed in tilapia head kidney cells. IL-$1\beta$ gene expression was analysed using QPCR (quantitative polymerase chain reaction). The ratio of the indicated gene expression was expressed as the relative mRNA level to $\beta$-actin mRNA level, which is constitutively expressed in macrophages. Consequently, head kidney cells incubated for three hours with rIL-$1\beta$(10, 2, 1 $\mu{g}$/ml) showed a dose dependent increase in IL-$1\beta$ mRNA levels and 1 $\mu{g}$/ml of poly I:C was also able to induce IL-$1\beta$ gene expression in head kidney in tilapia.

• Journal of Life Science
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• v.21 no.1
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• pp.159-164
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• 2011

Microbial whole-cell biosensors can be excellent analytical tools for monitoring environmental pollutants. They are constructed by fusing reporter genes (e.g., lux, gfp or lacZ) to inducible regulatory genes which are responsive to the relevant pollutants, such as aromatic hydrocarbons and heavy metals. A large spectrum of microbial biosensors has been developed using recombinant DNA technology and applied in fields as diverse as environmental monitoring, medicine, food processing, agriculture, and defense. Furthermore, their sensitivity and target range could be improved by modification of regulatory genes. Recently, microbial biosensor cells have been immobilized on chips, optic fibers, and other platforms of high-throughput cell arrays. This paper reviews recent advances and future trends of genetically modified microbial biosensors used for monitoring of specific environmental pollutants.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.966-971
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• 2009

Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the $Mut^s $ Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.

• Journal of Microbiology and Biotechnology
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• v.32 no.5
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• pp.551-563
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• 2022

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50℃ and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30℃, 40℃, and 50℃, respectively. The enzyme reserved its activity at 30℃ and 37℃ up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC₅₀ of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant Lasparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC₅₀ values of 1.53 and 18 IU, respectively.