Recombinant Plasmid Research Articles

Activation of ULK1 to trigger FUNDC1-mediated mitophagy in heart failure: Effect of Ginsenoside Rg3 intervention

BackgroundAlthough the development of therapies for heart failure (HF) continues apace, clinical outcomes are often far from ideal. Unc51-like-kinase 1 (ULK1)-mediated mitophagy prevents pathological cardiac remodeling and heart failure (HF). Molecularly ULK1-targeted agent to enhance mitophagy is scanty. Hypothesis/PurposeThis study aimed to investigate whether Ginsenoside Rg3 (Rg3) can activate ULK1 to trigger FUNDC1-mediated mitophagy for protecting heart failure. MethodsMolecular docking and surface plasmon resonance were used to detect the ULK1 binding behavior of Rg3. Established HF model in rats and transcriptome sequencing were used to evaluate the therapeutic effect and regulatory mechanism of Rg3. Loss-of-function approaches in vivo and in vitro were performed to determine the role of ULK1 in Rg3-elicited myocardial protection against HF. FUNDC1 recombinant plasmid of site mutation was applied to elucidate more in-depth mechanisms. ResultsStructurally, a good binding mode was unveiled between ULK1 and Rg3. In vivo, Rg3 improved cardiac dysfunction, adverse remodeling, and mitochondrial damage in HF rats. Furthermore, Rg3 promoted Ulk1-triggered mitophagy both in vivo and in vitro, manifested by the impetus of downstream Fundc1-Lc3 interaction. Of note, the protective effects conferred by Rg3 against mitophagy defects, pathological remodeling, and cardiac dysfunction were compromised by Ulk1 gene silencing both in vivo and in vitro. Mechanistically, Rg3 activated mitophagy by inducing ULK1-mediated phosphorylation of FUNDC1 at the Ser17 site, not the Ser13 site. ConclusionTogether these observations demonstrated that Rg3 acts as a ULK1 activator for the precise treatment of HF, which binds to ULK1 to activate FUNDC1-mediated mitophagy.

High-efficiency heterologous expression of nattokinase based on a combinatorial strategy

Nattokinase, traditionally produced by the fermentation of Bacillus natto, has a high-efficiency and safe thrombolytic effect. However, low activity limits its industrial application as a thrombolytic agent. In this study, the nattokinase encoding gene from Bacillus natto was heterologously expressed in B. subtilis WB800 to realize the high-efficiency expression. At first, the gene was amplified and connected to the Escherichia coli-B. subtilis shuttle plasmid pP43NMK to construct the recombinant plasmid pP43NMK-NKF. The enzyme activity increased to 75.3 FU/mL after the signal peptide starting codon was optimized as ATG from GTG. A total of 19 signal peptides were screened to construct a series of recombinants. The recombinant PSP7 showed the highest nattokinase enzyme activity, 235.2 FU/mL. Furthermore, constructing the promoter tandem expression vector proved that the dual-promoter recombinant PSP7C43 has the highest enzyme activity of 247.1 FU/mL. After the optimization of fermentation conditions, the nattokinase enzyme activity of recombinant PSP7C43 finally increased to 325.4 FU/mL. The combinatorial strategy adopted in this study realizes the substantial improvement of nattokinase expression level, which lays a foundation for the industrial development of nattokinase as a thrombolytic reagent.

Oral pcDNA3.1-VP4/VP56-FlaC DNA vaccine encapsulated by chitosan/sodium alginate nanoparticles confers remarkable protection against GCRV infection in grass carp

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) results in huge losses to the grass carp industry. In previous studies, DNA vaccination provided an efficient strategy for combating viral infection, but biodegradation and adverse environment hindered its application. In this study, Chitosan/sodium alginate (CS/SA) nanoparticles (NPs) loading system was applied to develop the oral DNA nano vaccine CS/SA/VP4C NPs and the oral DNA nano vaccine CS/SA/VP56C NPs to enhance its application effect. The recombinant plasmids pcDNA3.1-VP4C and pcDNA3.1-VP56C were constructed to express GCRV major outer capsid protein VP4 fusing Aeromonas hydrophila flagellin C (VP4C) and GCRV fiber protein VP56 fusing A. hydrophila flagellin C (VP56C), respectively. The oral DNA nano vaccines were about 100 nm in size with a regular spherical structure observed by transmission electron microscope. In addition, the fusion proteins VP4C and VP56C could be efficiently expressed in both fathead minnow cells and grass carp intestine epithelial cells by indirect immunofluorescence and western blotting analysis. The CS/SA/VP4C NPs and CS/SA/VP56C NPs were orally vaccinated with grass carp to evaluate the immune protective effect. The results showed that CS/SA/VP4C + VP56C NPs group had the highest protective effect that the survival rate increased by 42% compared with the control group. In addition, CS/SA/VP4C + VP56C NPs treatment could rapidly up-regulate serum innate immune indexes (total superoxide dismutase, lysozyme and complement C3), and significantly increase the mRNA expression of IFN1, MHC-IIα, IL-1β, TNFα, IgM, and IgT. Meanwhile, CS/SA/VP4C + VP56C NPs treatment greatly inhibited the replication of GCRV in the hindgut, spleen and head kidney. These results suggested that oral administration of CS/SA/VP4C + VP56C NPs could effectively prevent GCRV infection by improving adaptive immunity and innate immunity. Therefore, our study provides a new strategy for the fish viral disease prevention, which also establishes a theoretical basis for developing multiple DNA vaccines in the aquaculture industry.

Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury

BackgroundCerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI.MethodsSH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett’s T3 test (for unequal homogeneity) were used for statistical tests.ResultsERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1S616E or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed.ConclusionInhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model.9RDMMKPHSZ2No4hHf87VtDVideo

TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method.

Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5U of T5 exonuclease at 0°C for 5min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.

Soluble Expression of Antimicrobial Peptide BSN-37 from Escherichia coli by SUMO Fusion Technology.

Antimicrobial peptides (AMPs) are a kind of small molecular peptide that an organism produces to resist the invasion of foreign microorganisms. AMP BSN-37 is a bovine AMP that exhibits high antibacterial activity. In this paper, the optimized gene AMP BSN-37 was cloned into pCold-SUMO for fusion expression by recombinant DNA technology. The gene sequence of AMP BSN-37 was obtained by codons reverse translation, and the codons were optimized according to the codons preference of Escherichia coli (E. coli). The recombinant plasmid was constructed and identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transformed into BL21 E. coli to induce expression, and the IPTG concentration and time were optimized. The expressed soluble fusion protein SUMO-BSN-37 was purified by chromatography and then cleaved by SUMO proteases to release BSN-37. SDS-PAGE electrophoresis and Western blotting were used for identification. The recombinant plasmid pCold-SUMO-BSN-37 was obtained, and the fusion AMP BSN-37 was preliminarily expressed in BL21. After optimization, the optimal expression condition was 37 ℃ with 0.4 µM IPTG and 6h incubation. Under optimal conditions, a large amount of fusion AMP BSN-37 was obtained by purification. Western blotting showed that the fusion peptide was successfully expressed and had good activity. The expressed BSN-37 showed antimicrobial activity similar to that of synthesized BSN-37. In this study, soluble expression products of AMP BSN-37 were obtained, and the problem regarding the limited source of AMP BSN-37 could be effectively solved, laying a foundation for further research on AMP BSN-37.

Physical characteristics and stability profile of recombinant plasmid DNA within a film matrix

Plasmids are essential source material for production of biological drugs, vaccines and vectors for gene therapy. They are commonly formulated as frozen solutions. Considering the cost associated with maintenance of cold chain conditions during storage and transport, there is a significant need for alternative methods for stabilization of plasmids at ambient temperature. The objective of these studies was to identify a film-based formulation that preserved transfection efficiency of plasmids at 25 °C. A model plasmid, pAAVlacZ, was used for these studies. Transfection efficiency and agarose gel electrophoresis were utilized to assess bioactivity and changes in physical conformation of plasmid during storage. An amino acid, capable of sustaining a positive charge while supporting an alkaline environment within the film matrix, preserved transfection efficiency for 9 months at 25 °C. Addition of sugar and a plasticizer to the formulation preserved the plasmid in an amorphous state and improved handling properties of the film. The manner in which excipients were incorporated into bulk formulations and environmental humidity in which films were stored significantly impacted transfection efficiency of plasmid in the rehydrated solution. Taken together, these results suggest that plasmids can be stored for extended periods of time without refrigeration within a film matrix.

Immunogenicity Characterization of the Recombinant gI Protein Fragment from Pseudorabies Virus and an Evaluation of Its Diagnostic Use in Pigs.

Serological testing is an important method for the diagnosis of pseudorabies virus (PRV) infection. We aimed to investigate the envelope glycoprotein I (gI) of PRV, a strong immunogen, and its potential as an efficient and low-cost diagnostic reagent. In this study, the DNA of the PRV SC strain was used as the template, and the recombinant fragment of gI (633 bp) was amplified via PCR using synthetic primers, and was then ligated into the pET-30a expression vector. The constructs were transferred into Escherichia coli (E. coli) for prokaryotic expression, and the antigenicity of the expression products was identified by Western blot analysis with pig positive serum against PRV. The recombinant protein was purified by a Ni column, and BALB/c mice were immunized with purified gI protein to obtain anti-gI-positive serum. After PK-15 cells had been infected by PRV for 48 h, the immunogenicity of purified gI protein was identified with a fluorescence immunoassay using anti-gI mouse serum. The recombinant plasmid (pET-30a-gI) was expressed, and the native gI protein was obtained after denaturation by urea and renaturation by dialysis. A small-scale ELISA test containing 1.0 µg/mL of purified gI protein was designed to evaluate pig serum (80 samples), and the results of the ELISA test were compared to those of competitive ELISA (cELISA) tests using IDEXX Kits, which resulted in 97.5% consistency. The results suggested that the truncated gI protein may be a potential diagnostic reagent.

SlGSTE8 in Spodoptera litura participated in the resistance to phoxim and chlorpyrifos

AbstractGlutathione S‐transferases (GSTs) were reported to participate in insecticide resistance by metabolic and antioxidant activities. In our previous study, an ε class gene of GSTs, SlGSTe8 in Spodoptera litura, was screened out to be upregulated in a population resistant to pyrethroids and organophosphates. SlGSTe8 was highly expressed in the larvae stage, and the digestive tissue, foregut, midgut and hindgut, while the relative expression level was low in the pupae stage and other tissues. To further explore its role in the resistance to pyrethroids and organophosphates, the metabolic activity to insecticides by its recombinant protein was determined by Ultra Performance Liquid Chromatography, and its antioxidant enzyme activity was evaluated by disc diffusion assay. The recombinant protein showed significant metabolic activity to phoxim and chlorpyrifos, but not to fenvalerate, cyhalothrin or β‐cypermethrin. After incubation, the depletion rate of chlorpyrifos is 85.3%, higher than that of phoxim (17.5%). Also, the inhibition zone around filter discs decreased significantly after exposure to cumene hydroperoxide in recombinant plasmid than vector only, suggesting significant antioxidant activity of SlGSTE8. Further modelling and docking analysis indicated that the 3D structure of SlGSTE8 was well shaped for phoxim and chlorpyrifos, with the binding energy −5.58 and −5.15 kcal/mol, respectively. Our work provides evidence that SlGSTe8 in S. litura plays important roles in phoxim and chlorpyrifos resistance.

Screening and Identification of Dominant B-cell Epitopes of Human Cytomegalovirus UL138 and Its Clinical Applications

Aim: To predict the dominant B-cell epitope of the human cytomegalovirus UL138 gene and to provide a new assay for its latent infection. Methods: Recombinant plasmids were constructed with the predicted dominant epitopes, induced to be expressed and immunized in mice as a way to identify peptide antigenicity, immunogenicity and human serum. Results: Five potentially dominant B-cell epitopes were obtained. The positive rate of detection was found to be significantly higher in neonatal, adult and mouse serum specimens than in human cytomegalovirus (HCMV) (IgG, IgM) and UL138 gene DNA and cDNA assays (p < 0.05). Conclusion: The predicted dominant B-cell epitope is highly sensitive for the detection of human serum HCMV latent infection and can replace the traditional HCMV IgG assay.

Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae)

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA.

The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in Conus literatus. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in μ-PIIIA, three recombinant plasmids encoding different hybrid precursors of μ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of μ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in Escherichia coli, the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of μ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant μ-PIIIA without C-terminal amidate inhibited the current of hNaV1.4 with a 939 nM IC50. Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of μ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity.

Measuring Pathogenic Soil Fungi That Cause Sclerotinia Rot of Panax ginseng Using Real-Time Fluorescence Quantitative PCR

Sclerotinia ginseng is the primary pathogenic fungus responsible for Sclerotinia rot of ginseng, which significantly reduces plant yield and quality. The density of sclerotia in the soil is closely associated with rot incidence and severity. Whole genome sequencing was conducted to obtain fungal frame maps. The specific primers, q2001F/q2001R, were screened out by pan-genomic analysis using the NCBI database. Recombinant plasmids containing amplicons obtained with this primer set were used as standard plasmids to construct a real-time fluorescence quantitative PCR system. The relationships between the cycle threshold (Ct) values and the soil sclerotium densities were determined by real-time PCR. Real-time PCR had a detection limit of 1.5 × 10−2 g·kg−1 soil for Sclerotinia rot causing fungal mycelium, and the relationship between the density of S. ginseng mycelium n (g·g−1 soil) and the Ct value was n = 10(40.048 − Ct)/6.9541. The detection limit of real-time PCR for measuring soil sclerotia was 3.8 × 10−5 g·g−1 soil, suggesting a sensitivity 100 times that of conventional PCR. The relationship between the sclerotium density n (g·g−1 soil) and the Ct value was n = 10(18.351 − Ct)/7.0914. Compared with the conventional PCR method, the fluorescent quantitative PCR method could detect the population of Sclerotinia spp. in soil more efficiently, accurately, and sensitively.

Detection of Listeria monocytogenes in Food Using the Proofman-LMTIA Assay.

Microbial factors, including bacteria, viruses, and other pathogens, are significant contributors to foodborne illnesses, posing serious food safety risks due to their potential for rapid growth and contamination. Listeria monocytogenes is one of the most common types of foodborne bacteria that can cause serious foodborne diseases or even fatalities. In this study, a novel nucleic acid amplification method called Proofman-LMTIA was employed to detect Listeria monocytogenes contamination in food. This method combines proofreading enzyme-mediated probe cleavage with ladder-shape melting temperature isothermal amplification. A positive recombinant plasmid was used as a control to ensure the accuracy of the detection results, and primers and Proofman probes were specifically designed for the LMTIA. Genomic DNA was extracted, the reaction temperature was optimized, and the primers' specificity was verified using foodborne pathogens like Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella. The sensitivity was assessed by testing serial dilutions of genomic DNA, and the method's applicability was confirmed by detecting artificially contaminated fresh pork. The established LMTIA method exhibited both high specificity and sensitivity. At the optimal reaction temperature of 63 °C, the primers specifically identified Listeria monocytogenes contamination in pork at a concentration of 8.0 ± 0.7 colony-forming units (CFUs) per 25 g. Furthermore, the Proofman-LMTIA method was applied to test Listeria monocytogenes DNA in 30 food samples purchased from a Chinese retail market, and reassuringly, all results indicated no contamination. Proofman-LMTIA can serve as a reliable and rapid method for detecting Listeria monocytogenes in food, contributing to public health by safeguarding consumers from foodborne illnesses, and strengthening food safety regulations.

Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

Droplet digital PCR versus real-time PCR for in-house validation of porcine detection and quantification protocol: An artificial recombinant plasmid approach.

Authenticity and traceability are essential for modern food and medicine inspection, and reliable techniques are important for the trade of halal foods, which reach more than 20 percent of the world market. A sensitive and accurate porcine detection method is required to develop a conformity assessment system that includes laboratory testing for porcine-free certification. This study proposes a procedure that could be incorporated into the development of a standardized control and protocol for real-time PCR (qPCR) methods and their traceability using droplet digital PCR (ddPCR). The design used a recombinant pUC57 plasmid as an amplification target to carry the 97 bp fragment of the porcine ATCB gene. The absolute quantification and linearity assessment showed high precision with R2 values of 0.9971 and 0.9998 for qPCR and ddPCR, respectively. In general, both methods showed comparable results in terms of linearity and detection limit. However, both limit of detection assessments showed high sensitivity, although ddPCR showed a slightly higher sensitivity than that of qPCR, especially at low DNA concentrations. Multiple-sample and inter-participatory testing evaluations revealed a high sensitivity, broad applicability, and robustness of the qPCR method. Therefore, we conclude that based on a recombinant plasmid analysis with a low quantity (less than five copy number), the digital PCR method produced more reliable results. These results could provide scientific information for regulatory authorities, especially those in Indonesia, to consider the development and formulation of a well-established qPCR protocol for porcine detection using expected DNA concentrations.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO–K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea

Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His6-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl2, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. KdNTPd,app values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter kcat turned out to be 0.2 s−1 for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity.

Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis

In order to investigate the crucial role of ZmBT1 in starch accumulation during maize grain development and analyze the expression and distribution of ZmBT1 in various maize tissues, we prepared a polyclonal antibody. Specifically, we successfully expressed the recombinant plasmid pGEX-6p-ZmBT1-C (382-437aa) and purified Gst-ZmBT1-C as the antigen for antibody preparation. Our results confirmed that the ZmBT1 protein in maize tissues can be specifically recognized by the ZmBT1 antibody. Through Western blotting, we observed that the expression protein of ZmBT1 varied by tissues, with the highest content in the grain and endosperm. Furthermore, we employed a combination of Western blotting and quantitative real-time PCR to show that the expression level of ZmBT1 can be influenced by plant hormones. This finding suggests that ZmBT1 plays a critical role in the accumulation of starch and opens up new avenues for functional studies of this protein.

Combining isothermal recombinase polymerase amplification with lateral flow assay for diagnosis of P. cynomolgi malaria infection.

Plasmodium cynomolgi is a nonhuman primate parasite that causes malaria in humans and is transmitted by the Anopheles mosquito. Macaques, the natural hosts of P. cynomolgi, are widely distributed in Asia, especially in Southeast Asia. Anthropogenic land-use changes and wildlife habitat reduction due to local environmental changes, deforestation, urban expansion, and construction increased the frequency of human-macaque-vector interactions and facilitated the emergence of zoonotic malaria, causing an exponential increase in the infection rates in this area. Although microscopic tools are the gold standard for malaria diagnosis, they have very low sensitivity. Therefore, disease control and prevention require rapid, sensitive and accurate diagnostic tests. This study aims to develop a diagnostic method using a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method to specifically diagnose P. cynomolgi. Laboratory validation determined the method's sensitivity and specificity compared to the nested PCR method. The lower limit of detection was 22.14 copies/μl of recombinant plasmid per reaction. The combination method represented 81.82% sensitivity and 94.74% specificity compared to the nested PCR. The diagnostic testing developed in this study combines a recombinase polymerase amplification (RPA) and a lateral flow (LF) strip, offering rapid high sensitivity and specificity. Further development of this technique could make it a promising method for detecting P. cynomolgi.