Recombinant Expression Vector Research Articles

Expression of chicken epidermal growth factor (cEGF) in Escherichia coli regulates the microflora structure of the duodenum to improve growth performance and intestinal morphogenesis in broilers

ABSTRACT 1. A study used gene synthesis to obtain the functional domains of chicken epidermal growth factor (cEGF) and examined their impact on broiler growth performance, small intestinal morphology, digestive enzyme activities in the intestinal contents and the structure of duodenal microflora. 2. The pET-32a-cEGF recombinant expression vector was constructed. The specific band at 26 KDa was shown by SDS-PAGE analysis and WB results. The purified protein content was shown to be 1687 μg/ml by assay. 3. A total of 180 healthy, one-day-old Arbor Acres male, white-feathered broilers were randomly divided into three dietary treatment groups (six replicate pens, 10 birds per replicate): A control diet (ND); cEGF diet (cEGF), control supplemented with 250 mg/kg cEGF and the control diet (CD) supplemented with 250 mg/kg chlortetracycline. 4. The results showed that feeding the cEGF and CD diet reduced FCR of broilers aged 1–21 d, average daily feed intake (ADFI) at 22–42 d, and the FCR in the whole period (1–42 d; p < 0.05). Compared with the ND group, the cEGF diet increased duodenal α-amylase and alkaline phosphatase activities in the 1–21 d, duodenal lipase, alkaline phosphatase, and ileal alkaline phosphatase activities in the post-period and increased villus height in the duodenum and ileum (p < 0.05). In addition, the ACE and Chao1 index for the birds fed cEGF were higher than the ND group (p < 0.05). At the phyla level, Firmicutes and Proteobacteria were dominant in all groups. At the genus level, the dominant genus was Lactobacillus. The LEfSe analysis showed that the cEGF group was enriched by 11 species including Brevibacillus, Eisenbergiella, Cloacibacterium, Butyricoccus spp. 5. The addition of 250 mg/kg cEGF to the diet can increase growth performance by improving intestinal development and digestive enzyme activity, which may be related to the duodenal intestinal microflora. Therefore, cEGF is an effective alternative to antibiotics in broiler farming.

Heterologous expression and activity of α-helical antimicrobial peptide SW in Bacillus subtilis

The antimicrobial peptide Cecropin P1 (CP1) was isolated from the pig-intestinal parasitic nematode Ascaris lumbricoides and had potent antimicrobial activity. However, its cytotoxicity is high. Herein, Using CP1 as the parent peptide, we designed a derivative peptide SW by replacing amino acids based on the structure and function relationship of antimicrobial peptides (AMPs) to reduce the toxicity of CP1. Subsequently, the gene sequence encoding SacB-6 ×His-SUMO-SW was constructed into the recombinant expression vector PHY-P43 of Bacillus subtilis WB800N. The fusion proteins with concentrations of 26.97 mg/L were obtained after purification by a Ni–NTA column. A total of 5.37 mg of SW was obtained from 1 L fermentation cultures. Recombinant SW (rSW) exhibits potent antimicrobial activity against gram-negative bacteria by inducing membrane instabilities. In addition, rSW showed low hemolytic activity and cytotoxicity against human red blood cells and IPEC-J2, respectively. In the mice model of peritonitis infection, rSW could reduce Escherichia coli-loading in the liver, spleen, lung, and kidney of mice. These results indicate that rSW production by Bacillus. subtilis is a safe and reliable strategy, and rSW has the potential to be a novel antimicrobial agent against gram-negative bacterial infections.

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System.

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

Phenotypic and genotypic analysis of a patient with Miyoshi myopathy caused by truncated protein

Dysferlin protein deficiency can cause neuromuscular dysfunction, resulting in autosomal recessive dysferlinopathy, which is caused by DYSF gene mutation. Dysferlin proteins belongs to the Ferlin1-like protein family and are associated with muscle membrane repair and regeneration. In China, pathogenic mutations of the protein often result in two clinical phenotypes of Miyoshi muscular or limb band muscular dystrophy type 2B. It is clinically characterized by progressive muscle weakness and elevated serum creatine kinase. The data of the child were collected, blood samples of the child and his family members were collected, and whole exome sequencing (WES) was performed. The recombinant expression vector was constructed, the function of the mutation was verified by minigene, and the pathogenicity of the mutation was further analyzed by combining with biological information analysis. The patient initially presented with asymptomatic elevation of serum creatine kinase（CK）. Then progressive lower limb weakness, mainly distal limb weakness. Large amounts of scattered necrosis, myogenic lesions, and complete deletion of dysferlin protein were observed under muscle biopsy, which further improved genetic detection. Whole exome sequencing showed compound mutations (c.1397 + 1_1397 + 3del and c.1375dup p.M459Nfs*15) in DYSF gene. c.1375dup p.M459Nfs*15 have been reported. The other mutation is the deletion of c.1397 + 1_1397 + 3 in Intron15, which is an intron mutation that may affect splicing and the pathogenesis is still unknown. Minigene splicing assay verified that c.1397 + 1_1397 + 3del resulted in exon15 skipping and produced a premature termination codon. We report a novel pathogenic mutation in DYSF gene with Miyoshi myopathy and demonstrate this variant causes skipping of exon15 by minigene splicing assay. We point out the need of conducting functional analysis to verify the pathogenicity of intronic mutation. The finding enriches the mutation spectrum of DYSF gene and laid a foundation for future studies on the correlation between genotype and phenotype.

Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.).

Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants.

Effect of tandem repeats of antimicrobial peptide CC34 on production of target proteins and activity of Pichia pastoris

Antimicrobial peptides (AMPs) are attracting attention in the fields of medicine, food, and agriculture because of their broad-spectrum antibacterial properties, low resistance, and low-residue in the body. However, the low yield and instability of the prepared AMP drugs limit their application. In this study, we designed a tetramer of the AMP CC34, constructed and transfected two recombinant expression vectors with pGAPZαA containing a haploid CC34 and tetraploid CC34 (CC34-4js) into Pichia pastoris to explore the effect of biosynthesized peptides. The results showed that CC34 and CC34-4js expression levels were 648.2 and 1105.3 mg/L, respectively, in the fermentation supernatant of P. pastoris. The CC34-4js tetramer showed no antibacterial activity, could be cleaved to the monomer using formic acid, and the hemolytic rate of the polyploid was slightly lower than that of monomeric CC34. The average daily gain, average daily feed intake, feed conversion ratio and immune organ index of rats fed CC34 and CC34-4js showed no differences. In conclusion, CC34-4js exhibited a higher yield and lower hemolysis in P. pastoris than those of CC34. Finally, CC34 and CC34-4js enterokinase lysates showed similar antibacterial activity and both expressed peptides potentially improved the growth performance and organ indices of rats.

Cloning and functional characterization of a tau class glutathione transferase associated with haloxyfop-P-methyl resistance in Digitaria sanguinalis.

Haloxyfop-P-methyl, an acetyl-CoA carboxylase (ACCase)-inhibiting herbicide, has been extensively used to control grass weeds. Widespread use of haloxyfop-P-methyl in cotton fields in China has led to the development of glutathione transferase (GST)-mediated resistance in Digitaria sanguinalis. An RNA-seq analysis identified DsGSTU1, a tau class glutathione transferase from the D. sanguinalis transcriptome as a potential candidate. Here, we cloned DsGSTU1 from D. sanguinalis young leaf tissues and subsequently characterized DsGSTU1 by a combination of sequence analysis, as well as functional heterologous expression in rice. The full-length coding DNA sequence (CDS) of DsGSTU1 is 717 bp in length. Higher DsGSTU1 expression was observed in haloxyfop-P-methyl-resistant (HR) D. sanguinalis than in haloxyfop-P-methyl-susceptible (HS) plants. Overexpression of the DsGSTU1 gene was confirmed by transformation into the wild-type (WT) Nipponbare rice with pBWA(V)HS, a recombinant expression vector. GST activity in transgenic rice seedlings was 1.18-1.40-fold higher than the WT rice seedlings before and after haloxyfop-P-methyl treatment, respectively. Additionally, transgenic rice seedlings overexpressing DsGSTU1 were less sensitive to haloxyfop-P-methyl. Our combined findings suggest that DsGSTU1 is involved in metabolic resistance to haloxyfop-P-methyl in D. sanguinalis. A better understanding of the major genes contributing to herbicide-resistant D. sanguinalis facilitates the development of resistance management strategies for this global invasive grass weed. © 2023 Society of Chemical Industry.

A sensitive assay for scrutiny of Autographa californica multiple nucleopolyhedrovirus genes using CRISPR-Cas9.

Baculoviruses have very large genomes and previous studies have demonstrated improvements in recombinant protein production and genome stability through the removal of some nonessential sequences. However, recombinant baculovirus expression vectors (rBEVs) in widespread use remain virtually unmodified. Traditional approaches for generating knockout viruses (KOVs) require several experimental steps to remove the target gene prior to the generation of the virus. In order to optimize rBEV genomes by removing nonessential sequences, more efficient techniques for establishing and evaluating KOVs are required. Here, we have developed a sensitive assay utilizing CRISPR-Cas9-mediated gene targeting to examine the phenotypic impact of disruption of endogenous Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes. For validation, 13 AcMNPV genes were targeted for disruption and evaluated for the production of GFP and progeny virus - traits that are essential for their use as vectors for recombinant protein production. The assay involves transfection of sgRNA into a Cas9-expressing Sf9 cell line followed by infection with a baculovirus vector carrying the gfp gene under the p10 or p6.9 promoters. This assay represents an efficient strategy for scrutinizing AcMNPV gene function through targeted disruption, and represents a valuable tool for developing an optimized rBEV genome. KEY POINTS: [Formula: see text] A method to scrutinize the essentiality of baculovirus genes was developed. [Formula: see text] The method uses Sf9-Cas9 cells, a targeting plasmid carrying a sgRNA, and a rBEV-GFP. [Formula: see text] The method allows scrutiny by only needing to modify the targeting sgRNA plasmid.

Screening and Identification of Interacting Proteins of Mitfa in Red Tilapia

Skin color variation and differentiation are bottlenecks in the aquaculture industry of red tilapia. The mitfa gene primarily regulates melanocyte survival and proliferation in fish skin pigmentation. In this study, a recombinant expression vector for Mitfa expression was successfully constructed. After induction and purification, nearly 600 μg of Mitfa protein was purified from the fermentation supernatant. His pull-down assay, followed by LC-MS/MS analysis, revealed 10 proteins that interact with the Mitfa protein. Five of these proteins were annotated in the melanogenesis and tyrosine metabolism pathways, including Krt, Tpm, Myh, Tyr and Dct. The purified Mitfa recombinant protein was injected into red tilapia, and its activity and function were examined. The expression levels of the mitfa, tyr, tyrp1, and dct genes increased greatly in the dorsal skin after injection (p &lt; 0.05); meanwhile, the melanin content and tyrosinase activity increased significantly (p &lt; 0.05). These findings provide a theoretical basis for comprehending the function and regulatory mechanism of the red tilapia mitfa gene.

Construction of fluorescent transgenic zebrafish Tg (ttn.2: EGFP)

In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.

Production of Clubroot Standards Using a Recombinant Surrogate to Overcome Natural Genetic Variability.

Clubroot is caused by the obligate pathogen Plasmodiophora brassicae. The organism targets root hair cells for entry and forms spores in numbers so large that they eventually develop characteristic galls or clubs on the roots. Clubroot incidence is rising globally and impacting the production of oil seed rape (OSR) and other economically important brassica crops where fields are infected. P. brassicae has a wide genetic diversity, and different isolates can vary in virulence levels depending on the host plant. Breeding for clubroot resistance is a key strategy for managing this disease, but identifying and selecting plants with desirable resistance traits are difficult due to the symptom recognition and variability in the gall tissues used to produce clubroot standards. This has made the accurate diagnostic testing of clubroot challenging. An alternative method of producing clubroot standards is through the recombinant synthesis of conserved genomic clubroot regions. This work demonstrates the expression of clubroot DNA standards in a new expression system and compares the clubroot standards produced in a recombinant expression vector to the standards generated from clubroot-infected root gall samples. The positive detection of recombinantly produced clubroot DNA standards in a commercially validated assay indicates that recombinant clubroot standards are capable of being amplified in the same way as conventionally generated clubroot standards. They can also be used as an alternative to standards generated from clubroot, where access to root material is unavailable or would take great effort and time to produce.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Recombinant Production and Purification of Inosine 5-Mono Phosphate Dehydrogenase 1 Retinal Isoforms for Functional Studies

Inosine 5-monophosphate dehydrogenase 1 (IMPDH1) is the rate-limiting enzyme in the de novo purine nucleotide biosynthesis. IMPDH1 catalyzes IMP-oxidation to XMP, which in continue is converted to guanine nucleotides. Like mammals, the mouse IMPDH1 (mH1) has retinal-specific isoforms named H1 (546) and H1 (603). Mutations in the IMPDH1 gene are believed to cause retinal degenerative disease, retinitis pigmentosa, mediated by retinal-specific gene variants. After RNA extraction from the mouse retina, RT-PCR was done using NdeI and XhoI harboring primers. Tree mH1 isoform genes were amplified and cloned into a pET26b+ vector separately. The recombinant expression vectors were then transformed in E. coli BL21 (DE3) strain, expressed under IPTG-induced conditions and purified with Ni-NTA agarose resin. Activity assay of recombinant proteins was done by using spectrophotometric methods. Here, we cloned and optimized the expression and the purification of recombinant mH1 canonical and retinal isoforms in E. coli to gain soluble and highly active protein for further functional assays. Recombinant protein production in prokaryotic hosts, especially E. coli, is the most common method in large-scale of protein production for functional and structural studies. However, maximal yield and activity of recombinant proteins require optimal conditions for expression and purification, which is what we showed in the present study for the mouse IMPDH1 recombinant isoforms.

Secreting recombinant barnase by Lactococcus lactis and its application in reducing RNA from forages

Barnase is a ribonuclease used for plasmid purification, targeted gene therapy and studies of protein interactions. To make the use of barnase easier, the barnase gene from Bacillus amyloliquefaciens BH072 was cloned into Lactococcus lactis under the control of the PP5 or PnisA promoters. Four recombinant expression vectors were constructed with one or two signal peptides to control the enzyme secretion. 310 mg/L barnase was obtained in the presence of its inhibitor barstar after 36 h induction. The properties of barnase were investigated, showing that the optimal reaction temperature and pH were 50 °C and 5.0, respectively, and the highest enzyme activity reached 16.5 kU/mL. Barnase stored at 40 °C for 72 h retained 90 % of its initial activity, and maintained more than 80 % of its initial activity after 72 h of storage at pH 5.0–9.0. Furthermore, the optimal conditions for enzymatic reduction of nucleic acids in single-cell proteins (SCP) forages was investigated. 1 % salt solution with an SCP-enzyme ratio of 1000:1, pH 5.0 and incubated at 50 °C for 1 h, allowed 82 % RNA content reduction. Finally, homology modeling of barnase demonstrates its three-dimensional structure, and substrate simulation docking predicts key active residues as well as bonding patterns.

Structural characteristics of orexin receptor type 2 in Pacific abalone and its diurnal expression pattern after fasting and re-feeding

Pacific abalone (Haliotis discus hannai) is a typical nocturnal organism. To examine the circadian expression pattern of orexin receptor type 2 (OX2R) and its potential effect on the feeding behavior of abalone, the coding region sequence of OX2R that is 1215 bp in length and encodes 404 amino acids was first cloned using the rapid amplification of cDNA ends technique. A recombinant expression vector was constructed for H. discus hannai based on the OX2R protein, obtaining a recombinant protein with a molecular weight of 46 kDa. Polyclonal antibody was prepared with the purified recombinant protein used as the antigen, and the antibody titer of ≥512 K was detected by enzyme-linked immunosorbent assay. The expression levels of OX2R determined using western blotting were highest in the intestinal tract (P < 0.05), but they were not significantly different from the levels in the pedal. Immunofluorescence experiments affirmed that OX2R was widely expressed in the columnar cells of the intestinal mucosal epithelium. To further account for the relationship between the onset of feeding behavior and the expression level of OX2R in abalone, the circadian expression characteristics of OX2R were analyzed by dissecting the intestinal tissues after three days of normal feeding and fasting and following the refeeding treatment. The expression levels of OX2R in the refeeding group were significantly higher than those in the normal feeding and fasting groups at any time point (P < 0.05). The cosine curve analysis revealed that the expression levels of OX2R lost rhythmicity after fasting. Based on the quantification of behavioral data for abalone after fasting and refeeding, the cumulative movement distance and movement duration in each group followed a significant cosine rhythm (P < 0.05), which is consistent with abalone's nocturnal ecological habits. However, the cumulative movement distance and movement duration in the fasting group were significantly lower than those in the normal feeding and refeeding groups (P < 0.05). The peak phases of the cumulative movement distance and movement duration in the refeeding group (ZT08:22 and ZT08:44) shifted backward compared to the normal feeding group (ZT07:33 and ZT07:39). The above results first identified the structural characteristics and circadian expression patterns of OX2R in the marine mollusk abalone, which may reveal the molecular mechanism behind the generation of a feeding rhythm in marine nocturnal organisms and serve as a tool helping to maintain the diversity of marine benthic resources.

Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML.

We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 109 and 2.61 × 109IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.

Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies

Fibroblasts are the main cells of connective tissue and have pivotal roles in the proliferative and maturation phases of wound healing. These cells can secrete various cytokines, growth factors, and collagen. Vascular endothelial growth factor (VEGF) is a unique factor in the migration process of fibroblast cells through induces wound healing cascade components such as angiogenesis, collagen deposition, and epithelialization. This study aimed to create VEGF165 overexpressing fibroblast cells to evaluate angiogenesis function in wound healing. In vitro, a novel recombinant expression vector, pcDNA3.1(-)-VEGF, was produced and transfected into the fibroblast cells. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying and qualifying methods. Mechanical, physical, and survival properties of polyurethane-cellulose acetate (PU-CA) scaffold were investigated. Finally, in vivo, the angiogenic potential was evaluated in four groups containing control, PU-CA, PU-CA with fibroblast cells, and VEGF-expressing cells on days 0, 2, 5, 12 and 15. Wound biopsies were harvested and the healing process was histopathologically evaluated on different days. qRT-PCR showed VEGF overexpression (sevenfold) in genetically-manipulated cells compared to fibroblast cells. Recombinant VEGF expression was also confirmed by western blotting. Manipulated fibroblast cells represented more angiogenesis than other groups on the second day after surgery, which was also confirmed by the antiCD31 antibody. The percentage of wound closure area on day 5 in genetically-manipulated Hu02 and Hu02 groups showed a significant reduction of wound area compared to other groups. These findings indicate that overexpression of VEGF165 in fibroblast cells results in enhanced angiogenesis and formation of granulated tissue in the early stage of the healing process, which can show its therapeutic potential in patients with impaired wound healing and also provide functional support for gene therapy.

The molecular and functional characterization of ferritins in the hard tick Hyalomma rufipes

BackgroundThe protein ferritin, which plays an important role in the maintenance of iron homeostasis, is indispensable for iron detoxification, resistance to oxidative stress and innate immunity. Ticks, which are obligate blood-sucking ectoparasites, have to deal with a large amount of iron when they take a blood meal.MethodsSequence analysis was undertaken using bioinformatics. A recombinant (r) expression vector, rferritin, was constructed for a prokaryotic expression system. A quantitative polymerase chain reaction platform was used to detect the spatial and temporal expression patterns of target genes and their responses to a low temperature environment. Knockdown of the ferritin genes through RNA interference was used to analyze their effects on physiological parameters of ticks.ResultsTwo ferritin genes, HrFer1 and HrFer2, were cloned from the tick Hyalomma rufipes. Their open reading frames are 519 base pairs (bp) and 573 bp in length, and number of coding amino acids 170 and 190, respectively. The phylogenetic tree showed that HrFer1 and HrFer2 have a close evolutionary relationship with the H subunit of ferritin. In vitro experiments showed that rHrFer1 and rHrFer2 had concentration-dependent iron chelating activity. The relative expression of the two ferritin genes was higher in the ovary and midgut of H. rufipes. RNA interference results demonstrated that HrFer1 and HrFer2 expression had a significant effect on engorged body weight, number of eggs laid, and mortality of H. rufipes, and that HrFer2 also had a significant effect on feeding duration. Furthermore, the relative expression of ferritin decreased significantly in a low temperature environment, suggesting that HrFer1 and HrFer2 play a regulatory role in the cold stress response of H. rufipes.ConclusionsThe results of the present study improve our understanding of the involvement of ferritins in tick blood-feeding.Graphical

Subunit vaccine based on glycoprotein B protects pattern animal guinea pigs from tissue damage caused by infectious bovine rhinotracheitis virus

Infectious bovine rhinotracheitis (IBR) is caused by Bovine herpesvirus type 1 (BoHV-1), which seriously threatens the global cattle industry. Only vaccination to improve immunity is the most direct and effective means to prevent IBR. Attempts are being made to use subunit vaccines, deleted or recombinant viral vaccines to reduce or eradicate IBR. For investigating the immunological characteristics of glycoprotein B subunit vaccine in pattern animal guinea pigs, the partial glycoprotein B (gB) of BoHV-1 with dominant antigenic characteristic was selected. A recombinant prokaryotic expression vector pET-32a-gB with the truncated gB gene was constructed, expressed, identified and the purified proteins were used to immunize guinea pigs. The immune effect of the subunit vaccine was assessed by monitoring clinical symptoms, viral load, antibody secretion, and histopathological changes. The results indicated that guinea pigs immunized with the gB subunit vaccine produced high levels of anti-gB antibodies and virus-neutralizing antibodies. The gB subunit vaccine significantly reduced viral shedding and lung tissue damage after IBRV challenge. The animals inoculated the gB subunit vaccine also had less virus reactivation. Its protective effect on viral shedding and tissue damage was similar to that of inactivated BoHV-1 vaccine. This work is a proof-of-concept study of subunit vaccine-induced protection against BoHV-1. And it is expected to be a candidate vaccine for the prevention of IBR.