Recombinant Clones Research Articles

Classical Recombinant DNA Cloning.

Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a gene. The main purpose of cloning is to study either that DNA sequence or the RNA or protein product it encodes. Building on key enzymatic discoveries in the late 1960s, gene cloning was pioneered in the early 1970s. Since then, DNA cloning and manipulation have been used in every area of biological and biomedical research, from molecular genetics, structural biology, and developmental biology to neurobiology, ancient DNA studies, and immunology. It is a versatile technique that can be applied to a variety of starting DNA types and lengths, including cDNAs, genes, gene fragments, chromosomal regions, or shorter fragments such as PCR products and functional control regions such as enhancers or promoters. The starting DNA can originate from any cell, tissue, or organism. In this chapter we will cover traditional ("classic") molecular cloning strategy. This comprises six linked stages in which (1) PCR is used to amplify a DNA region of interest that is then (2) digested with restriction enzymes, alongside a selected vector, to produce complementary ends crucial for the two molecules to be (3) ligated by an ATP-dependent DNA ligase, creating a recombinant DNA molecule. The recombinant DNA is then (4) introduced into competent bacterial cells by transformation and (5) grown on a selective agar media, followed by (6) colony-PCR for screening purposes. We provide a worked example to demonstrate the cloning of an average-size gene (in this case the 2kb DNA ligase A gene) from E. coli into a common plasmid expression vector. We have included six color figures and two tables to depict the key stages of a classical molecular cloning protocol. If you are cloning a segment of DNA or a gene, remember that each DNA cloning experiment is unique in terms of sequence, length, and experimental purpose. However, the principles of traditional cloning covered in this chapter are the same for any DNA sequence; we have included a detailed notes section, so you should easily be able to transfer them to your own work. Some of the following chapters in this volume will cover other, more recently developed, cloning protocols.

Identification of antibody against wingless‑type MMTV integration site family member 7B as a biliary cancer tumor marker.

Biliary cancer has a poor prognosis due to a lack of specific biomarkers and difficulty in diagnosis. The present study aimed to identify serum tumor markers for the diagnosis of biliary cancer via serological identification of antigens by recombinant cDNA expression cloning. Wingless‑type MMTV integration site family, member 7 (WNT7B) was identified as a target antigen, suggesting the presence of serum antibodies against this antigen. Deletion mutants were then prepared to evaluate the response to serum antibodies. When serum antibody levels against WNT7B deletion mutants (WNT7B-92‑2, -92‑260, 2-260 and 184-260) were examined using amplified luminescence proximity homogeneous assay‑linked immunosorbent assay, the levels of the antibody against WNT7B with amino acids 184‑260 were higher in patients with biliary cancer than in healthy donors. Therefore, the region covering residues 184‑260 of WNT7B was decomposed to generate seven peptides, and the levels of antibodies against these peptides were measured. Among them, the levels of antibodies against WNT7B234‑253 and WNT7B244‑260 were higher in patients with biliary cancers than in healthy donors (WNT7B234‑253, P=0.0009; WNT7B244‑260, P=0.0005). The levels of the antibody against the former were specifically high in patients with biliary cancer but not in those with esophageal, gastric, colorectal, pancreatic, or breast cancer. Furthermore, analysis by the cutoff value of WNT7B234‑253 defined by ROC showed a high sensitivity of 70% in patients with biliary cancer. Therefore, the serum levels of the antibody against WNT7B234‑253 may be useful as a marker for biliary cancer diagnosis.

Short Communication: Cloning, production and responses of the sMTL-13 protein (13 kDa lectin secretion) Mycobacterium tuberculosis against Interleukin 6

Abstract. Agus R, Purnamasari WOS, Parura RD, Silang F, Massi MN. 2022. Short Communication: Cloning, production and responses of the sMTL-13 protein (13 kDa lectin secretion) Mycobacterium tuberculosis against Interleukin 6. Biodiversitas 23: 6208-6212. The Bacillus Calmette-Guérin (BCG) vaccine protected effect against meningitis and disseminated Tuberculosis TB in children. However, it does not prevent primary infection and reactivation of latent pulmonary infection. Therefore, an effective vaccine is needed to prevent tuberculosis. One of the potentials is the sMTL-13 protein (13 kDa lectin secretion), which causes an increase in interleukin 6 (IL-6) as a proinflammatory cytokine and shows high titers in the serum of TB patients. This study aimed to clone, express and evaluate the sMTL-13 recombinant protein. The research steps were ligation of Rv1419 to pQE-30Xa, transformation to Escherichia coli BL-21, and production of recombinant protein with IPTG induction. Furthermore, immunoreactivity tests with the serum of TB patients and healthy serum using ELISA. The results showed that the PCR product Rv1419 showed a band of 474 bp and white colonies as a recombinant clone of pQE-30Xa-Rv1419. The production of sMTL-13 recombinant protein has been successfully carried out and produces 13 kDa protein. The results of the immunoreactivity test showed that the serum level of IL-6 in patients with active TB was 84.22 pg/mL and the serum level of IL-6 in healthy people was 63.74 pg/mL.

Biotechnology: use of available carbon sources on the planet to generate alternatives energy

Biotechnology has been an essential tool in the search for solutions and in the optimization of bioprocesses associated with issues of human, plant, animal, energy and also the balance of ecosystems on planet Earth. The objective of this research was to present an unconventional substrate (cellulose), in abundance on the planet, to be used as a substitute source of carbon and energy for biotechnology processes, with the possibility of increasing industrial production of biomass and energy. As basis for the research, an extensive literature review and quantitative and qualitative analyzes were carried out. Genetic Engineering techniques were used to enable the yeast Saccharomyces cerevisiae for partial cellulose degradation, through the use of genetic transformation methods to insert a plasmid carrying the cellobiohydrolase cDNA. It was found that the recombinant and biologically active cellobiohydrolase protein was expressed and excreted in haploid and diploid laboratory yeast strains. The analyzes allowed the visualization of cellulolysis halos around colonies of recombinant strains grown in solid YPD medium with 1% microgranular cellulose. The recombinant clones derived from the haploid lineage yielded in average of 1.70 mg ART/mL, while recombinant clones derived from the diploid lineage produced in average of 2.05 mg ART/mL.

Characterization and identification of a novel chromosomal class C β-lactamase, LAQ-1, and comparative genomic analysis of a multidrug resistance plasmid in Lelliottia amnigena P13.

Lelliottia amnigena, a bacterium usually isolated from natural environments, may cause human infections and has been suggested to be naturally resistant to second- and third-generation cephalosporins. In this study, we determined the whole-genome sequence of an isolate, L. Amnigena P13, isolated from animal farm sewage. On the basis of genome sequence analysis, susceptibility testing, molecular cloning, and enzyme kinetic parameter analysis, we identified a novel chromosome-encoded AmpC β-lactamase, LAQ-1. bla LAQ-1 is resistant to penicillin G, ampicillin, and several first- to fourth-generation cephalosporins, such as cefazolin, cefoxitin and cefepime. The MIC levels of some β-lactams, such as cefoxitin, cefepime, aztreonam and cefazolin, for the recombinant clone (pUCP24-bla LAQ-1/DH5α) increased by approximately 4- to 64-fold compared with those of the control strain (pUCP24/DH5α). The kinetic properties of LAQ-1, with the highest catalytic activity observed toward piperacillin, were basically the same as those of typical class C β-lactamases, and avibactam had a strong inhibitory effect on its hydrolytic activity. The genetic background of bla LAQ-1 was relatively conserved, and no mobile genetic element (MGE) was found around it. The plasmid pP13-67 of L. amnigena P13 harbored 12 resistance genes [qnrS1, aph(6)-Id, aadA2, sul1, sul2, bla TEM-1, qacEΔ1, dfrA12, tetA and floR] related to different mobile genetic elements within an ~22 kb multidrug resistance region. The multidrug resistance region shared the highest nucleotide sequence similarities with those of the chromosomes or plasmids of different bacterial species, indicating the possibility of horizontal transfer of these resistance genes among different bacterial species.

Severinia buxifolia-isolated acridones inhibit lung cancer invasion and decrease HIFα protein synthesis involving 5’UTR-mediated translation inhibition

BackgroundLung cancer is one of the most common cancers worldwide and is by far the leading cause of cancer death attributed to its rapid metastasis and poor prognosis. Given that hypoxia-inducible factors (HIFs) are associated with cancer metastasis, discovering agents to inhibit HIF-mediated invasive cancer is highly desired. PurposeThis study aimed to investigate the natural acridone compounds isolated from Severinia buxifolia for the potential to delay hypoxia-induced lung cancer invasiveness by HIF inhibition. MethodsUsing a hypoxia-responsive element (HRE) luciferase reporter, cell migration and invasion assays, real-time PCR, Western blot, and DNA recombinant clones, compound effect on HIF activity, cancer metastasis, HIF-1α mRNA transcription, HIFs protein stability, and HIF-1α translation were observed under hypoxia conditions. ResultsAtalaphyllidine (Sbs-A) and atalaphyllinine (Sbs-B) were found to show the most potent effects on HIF transcriptional activity and HIF-1α protein expression in NSCLC cell line A549, although Sbs-A and Sbs-B might not attribute decreasing HIF-1α mRNA expression to potent inhibition of HIF activity. HIF-1α protein stability was not affected by Sbs-A; also, prolyl hydroxylase and proteasome inhibitors could not reverse the inhibitory effect from compounds. Furthermore, 3 - 10 μM low concentrations of Sbs-A inhibited HIF target gene expression, gelatin zymography activity, and A549 cancer invasion. Ultimately, Sbs-A inhibited HIF-1α 5′UTR-mediated translation independent of oxygen concentration, underlying the mechanism of compounds inhibiting HIF-1α protein expression. ConclusionOur study proposed Severinia buxifolia-isolated acridone compounds inhibited 5’-mRNA HIFA-mediated translation and provided evidence supporting the ability of acridone compounds in targeting HIFα for delayed lung cancer metastasis.

Cytotoxic effect of cloned EGFP gene on NCI-H727 cell line via genetically engineered gene transfer system

Introduction and Aim: Cancers are a complex group of genetic illnesses that develop through multistep, mutagenic processes which can invade or spread throughout the body. Recent advances in cancer treatment involve oncolytic viruses to infect and destroy cancer cells. The Newcastle disease virus (NDV), an oncolytic virus has shown to have anti-cancer effects either directly by lysing cancer cells or indirectly by activating the immune system. The green fluorescent protein (GFP) has been widely used in studying the anti-tumor activity of oncolytic viruses. This study aimed to study the anticancer effect of a recombinant rNDV-GFP clone on NCI-H727 lung carcinoma cell line in vitro. Materials and Methods: The GFP gene was inserted to a NDV strain to create a recombinant NDV (rNDV- GFP) using reverse genetics technology. The MTT assay was used in evaluating the oncolytic effect of rNDV- GFP on the lung carcinoma NCI-H727 cells. Light and fluorescent microscopy was used to study the cytopathic effects of rNDV-GFP. Results: MTT assay showed that rNDV-GPF inhibited the NCI-H727 tumor cell death in a time-dependent manner. A significant inhibitory effect (78.3%) for rNDV-GPF on cancer cells was observed at 96h in comparison to rNDV (22.7%) and the cytotoxicity rate was directly proportional to the MOI used. Microscopic studies showed rNDV-GPF to induce cytopathic effect post 24 h of infection. Conclusion: The GFP-expressing recombinant NDV strains exhibited encouraging results in terms of tumor growth inhibition. Our research set the groundwork for employing recombinant NDV as an anticancer viral vector.

Optimization of haloacid dehalogenase production by recombinant E. coli BL21 (DE3)/pET-hakp1 containing haloacid dehalogenase gene from Klebsiella pneumoniae ITB1 using Response Surface Methodology (RSM)

Organohalogens, including monochloroacetic acid (MCA), are abundantly synthesized compounds for various industrial purposes. MCA is widely used as a raw material or as an intermediate compound for the production of pesticides, herbicides, fungicides, plastics, surfactants, shampoos, liquid soaps, and emulsion agents. Nonetheless, widespread and large-scale utilization of organohalogens might negatively impact life quality as these compounds are toxic to organisms and persistently present in the environment. An effort to decrease the effect of MCA pollutant is by performing bioremediation, taking advantage of microorganisms that produce haloacid dehalogenases, a class of enzymes that catalyze the breakage of carbon halogen bonds. In this sense, we have isolated Klebsiella pneumoniae ITB1 that could degrade MCA. The haloacid dehalogenase gene from this bacterium has been successfully cloned into pGEM-T vector and subcloned into pET-30a(+) expression vector to yield pET-hakp1 recombinant clone in Escherichia coli BL21 (DE) host cell. This research aimed to find an optimum condition for producing haloacid dehalogenase from this recombinant clone using Response Surface Methodology (RSM). Among the independent variables studied were the concentration of inducer, incubation temperature after the induction, and incubation period after the induction. We obtained the crude extract of the enzyme as cells' lysate after sonicating the bacterial cells. Haloacid dehalogenase activity against MCA substrate was determined by measuring the amount of chloride ions released into the medium of the enzymatic reaction using the colorimetry method, according to Bergmann and Sanik. The result indicated that the optimum condition for haloacid dehalogenase production by E. coli BL21 (DE3)/pET-hakp1 was observed when using 1.8 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside) as the inducer, followed by 4 h incubation with shaking at 37 °C, which was predicted to result in a maximum of 0.48 mM chloride ions from 0.50 mM of MCA substrate. This report provides an insight into applying RSM for optimization of enzyme production from E. coli recombinant clones.

Diversity of bacterial phenol hydroxylase-encoding genes from gasoline-contaminated silt soil

Abstract. Vasandani SR, Tan WA. 2022. Diversity of bacterial phenol hydroxylase-encoding genes from gasoline-contaminated silt soil. Biodiversitas 23: 5502-5506. Phenol is an aromatic compound often used as a raw material or intermediate in various industries. Improper handling and disposal may lead to the accumulation of this hazardous compound. Bioremediation is the most viable method to remove phenol from contaminated environment. In this study, the diversity of genes that encode for phenol hydroxylase, a key enzyme in phenol degradation, was assessed in gasoline-contaminated soil from a commercial gas station in Central Jakarta, Indonesia. Partial phenol hydroxylase-encoding gene library was constructed using a pair of universal primer in the pGEM®-Teasy vector. A total of 30 recombinant clones were obtained and sequenced to analyze the genetic diversity of this gene. Obtained clones were 86-99% identical to phenol hydroxylase-related proteins. Phylogenetic tree analysis on amino acid sequences derived from our library revealed that 56.7% of the cloned fragments were closely related to Proteobacteriodota and 20.0% of them were clustered with Actinomycetota. The rest 23.3% of the clones formed a cluster separate from any of the reference sequences, possibly indicating the presence of novel phenol hydroxylase genes.

Development of a Next-Generation Vaccine Platform for Porcine Epidemic Diarrhea Virus Using a Reverse Genetics System.

For the past three decades, the porcine epidemic diarrhea virus (PEDV) has remained an enormous threat to the South Korean swine industry. The scarcity of an effective method for manipulating viral genomes has impeded research progress in PEDV biology and vaccinology. Here, we report the development of reverse genetics systems using two novel infectious full-length cDNA clones of a Korean highly pathogenic-G2b strain, KNU-141112, and its live attenuated vaccine strain, S DEL5/ORF3, in a bacterial artificial chromosome (BAC) under the control of a eukaryotic promoter. Direct transfection of cells with each recombinant BAC clone induced cytopathic effects and produced infectious progeny. The reconstituted viruses, icKNU-141112 and icS DEL5/ORF3, harboring genetic markers, displayed phenotypic and genotypic properties identical to their respective parental viruses. Using the DNA-launched KNU-141112 infectious cDNA clone as a backbone, two types of recombinant viruses were generated. First, we edited the open reading frame 3 (ORF3) gene, as cell-adapted strains lose full-length ORF3, and replaced this region with an enhanced green fluorescent protein (EGFP) gene to generate icPEDV-EGFP. This mutant virus presented parental virus-like growth kinetics and stably retained robust EGFP expression, indicating that ORF3 is dispensable for PEDV replication in cell culture and is a tolerant location for exogeneous gene acceptance. However, the plaque size and syncytia phenotypes of ORF3-null icPEDV-EGFP were larger than those of icKNU-141112 but similar to ORF3-null icS DEL5/ORF3, suggesting a potential role of ORF3 in PEDV cytopathology. Second, we substituted the spike (S) gene with a heterologous S protein, designated S51, from a variant of interest (VOI), which was the most genetically and phylogenetically distant from KNU-141112. The infectious recombinant VOI, named icPEDV-S51, could be recovered, and the rescued virus showed indistinguishable growth characteristics compared to icKNU-141112. Virus cross-neutralization and structural analyses revealed antigenic differences in S between icKNU-141112 and icPEDV-S51, suggesting that genetic and conformational changes mapped within the neutralizing epitopes of S51 could impair the neutralization capacity and cause considerable immune evasion. Collectively, while the established molecular clones afford convenient, versatile platforms for PEDV genome manipulation, allowing for corroborating the molecular basis of viral replication and pathogenesis, they also provide key infrastructural frameworks for developing new vaccines and coronaviral vectors.

Bioengineering and computational analysis of programmed cell death ligand-1 monoclonal antibody.

The trans-membrane proteins of the B7 family programmed cell death ligand-1 (PD-L1) and programmed death-1 (PD-1) play important roles in inhibiting immune responses and enhancing self-tolerance via T-cell modulation. Several therapeutic antibodies are used to promote T-cell proliferation by preventing interactions between PD-1/PD-L1. Recombinant technology appears to be quite useful in the production of such potent antibodies. In this study, we constructed recombinant molecules by cloning variable regions of the PD-L1 molecule into pMH3 vectors and transferring them into mammalian cell lines for expression. G418 supplementation was used to screen the recombinant clones, which were then maintained on serum-free medium. The full-length antibody was isolated and purified from the medium supernatant at a concentration of 0.5-0.8 mg/ml. Antibody binding affinity was investigated using ELISA and immunofluorescence methods. The protein-protein interactions (PPI) were determined using a docking approach. The SWISS model was utilized for homology modeling, while ZDOCK, Chimera, and PyMOL were used to validate 3D models. The Ramachandran plots were constructed using the SWISS model, which revealed that high-quality structures had a value of more than 90%. Current technologies allow for the accurate determination of antigen-antibody interactions.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

SummaryLiver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus.

DNA purification methods are indispensable tools of molecular biology, used for many decades. Nevertheless, for certain specialized applications, the currently employed techniques are not sufficiently effective. While examining a number of the existing methods to purify the genomic DNA of the thermophilic bacteriophage TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus), we have found out that the obtained DNA is contaminated with trace amounts of infectious TP-84 particles. This was detrimental for the bacteriophage genetic manipulation purposes, as finding the recombinant TP-84 clones was essentially impossible due to the appearance of a high background of native bacteriophage plaques. Thus, we have developed a method, which enables the fast and efficient isolation of a bacteriophage genomic DNA from concentrated phage preparations, obtained using CsCl gradient ultracentrifugation, without the need to remove concentrated CsCl solutions. The method employs silica columns and mini-scale isolation of microgram amounts of high quality DNA. It is universal—the silica mini-columns from various manufacturers can be used to conduct the procedure. The purified DNA, free from infectious bacteriophage particles, is ready for further manipulations. This is particularly important for such thermophilic bacteriophages that may partially survive standard isolation procedures and contaminate the final DNA product.

First Report of Tobacco Ringspot Virus Naturally Infecting Cotton (Gossypium hirsutum L.) in the United States.

Cotton (Gossypium hirsutum L.) is one of the major cash crops grown in the United States (U.S.) with a total acreage of over 11.5 million acres in 2021 (NASS, 2021). In Oklahoma, cotton represents an important economic crop and was grown on 490,000 acres during the 2021 growing season (NASS, 2021). In 2021, during a survey of a cotton field in Beckham County of Oklahoma, cotton plants showed typical virus-like symptoms including mosaic, yellow ring spots, discoloration and short internodes (Supplementary Fig. 1). Thirteen symptomatic and five asymptomatic samples were collected from cotton plants and brought to the University of Tulsa for further processing. Total RNA was extracted from all samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich). Total RNA from two symptomatic samples (named EC3 and EC4) were subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 High-Output kit v2.5 (Illumina, USA) at the genomic facility, Oklahoma State University (Stillwater, OK, USA). A total of 17,542,322 and 22,572,118 trimmed pair-ends reads for both samples were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc) and subjected to BLASTn analysis. Two contigs of 556 bp and 1062 bp (average coverage 2,799X) for sample EC3 showed 99% and 91% nucleotide (nt) identities with 3'-UTR, 5'-UTR and P1A gene respectively of the Tobacco ringspot virus (TRSV) RNA1 of isolates WA-AM1 (MW495243.1), and IA-1-2017 (MT563078.1) respectively. The other two contigs, 221 bp and 561 bp (average coverage 23,070X) for sample EC4 showed, 100% and 96% nt identities with 3'-UTR, protease, and RdRp genes of TRSV RNA1, isolates WA-AM1 and YW (MT042825.1) respectively. The HTS data did not reveal any other viral sequence in these two cotton samples. To further confirm the presence of TRSV in these samples, previously designed specific primers to TRSV (Forward: 5'-GGAAATTAACTGGGATGATTT-3' and Reverse: 5'-GAGCTCCAACCTTAAAACCA-3') for RNA1, targeting the P1A and helicase genes, and another primer pair (Forward: 5'-GCATCCTCCCATGTTTTCT-3' and Reverse: 5'-GGGACAAACACGACACTA-3') for RNA2, targeting the coat protein and 3'-untranslated region, of TRSV were tested by RT-PCR assay. The sizes of amplified PCR products obtained from both isolates (EC3 and EC4) on 1% agarose gel were approximately 1,000 bp for RNA1 and 1,100 bp for RNA2. The amplified PCR products were cloned and three independent recombinant clones for each primer set were analyzed by Sanger sequencing. The resulting consensus sequences were used in a BLASTn search against the Genbank and matched with TRSV sequences. Consensus nt sequences analysis specific to RNA1 of EC3 isolate (Accession no. OM563300) and EC4 isolate (Accession no OM563301) showed 97% nt identities with TRSV isolate IA-1-2017. Consensus nt sequences specific to RNA 2 of EC3 isolate (Accession no. OM630605) and EC4 isolate (Accession no OM5630606) showed 92% and 90% nt identities with the corresponding sequences of WA-AM1 and IA-1-2017 isolates respectively. Further screening of the remaining 11 symptomatic samples resulted in two more positive TRSV samples that were co-infected with Cotton leafroll dwarf virus (CLRDV), six were positive to only CLRDV, and three were negative to both viruses by RT-PCR assay using the above TRSV specific primers and CLRDV specific primers AL674F/1407R (Avelar et al. 2019). None of the asymptomatic cotton samples were positive by RT-PCR to TRSV or CLRDV. Our results confirmed the presence of TRSV infection in these symptomatic cotton plants. The presence of TRSV could pose a new threat to cotton crops in Oklahoma and the U.S. due to its wide host range (Adam and Antoniw, 2005), and transmission through many vectors including nematodes, (Keinath et al. 2017), and seed (Hill and Whitman, 2014). To the best of our knowledge, this is the first report of TRSV infecting cotton naturally in the U.S. and in the world.

Productive Replication of HIV-1 but Not SIVmac in Small Ruminant Cells.

Animal lentiviruses (LVs) have been proven to have the capacity to cross the species barrier, to adapt in the new hosts, and to increase their pathogenesis, therefore leading to the emergence of threatening diseases. However, their potential for widespread diffusion is limited by restrictive cellular factors that block viral replication in the cells of many species. In previous studies, we demonstrated that the restriction of CAEV infection of sheep choroid plexus cells was due to aberrant post-translation cleavage of the CAEV Env gp170 precursor. Later, we showed that the lack of specific receptor(s) for caprine encephalitis arthritis virus (CAEV) on the surface of human cells was the only barrier to their infection. Here, we examined whether small ruminant (SR) cells can support the replication of primate LVs. Three sheep and goat cell lines were inoculated with cell-free HIV-1 and SIVmac viral stocks or transfected with infectious molecular clone DNAs of these viruses. The two recombinant lentiviral clones contained the green fluorescent protein (GFP) reporter sequence. Infection was detected by GFP expression in target cells, and the infectious virus produced and released in the culture medium of treated cells was detected using the indicator TZM-bl cell line. Pseudotyped HIV-GFP and SIV-GFP with vesicular stomatitis virus G glycoprotein (VSV-G) allowed the cell receptors to be overcome for virus entry to further evaluate the viral replication/restriction in SR cells. As expected, neither HIV nor SIV viruses infected any of the SR cells. In contrast, the transfection of plasmid DNAs of the infectious molecular clones of both viruses in SR cells produced high titers of infectious viruses for human indicators, but not SR cell lines. Surprisingly, SR cells inoculated with HIV-GFP/VSV-G, but not SIV-GFP/VSV-G, expressed the GFP and produced a virus that efficiently infected the human indictor, but not the SR cells. Collectively, these data provide a demonstration of the lack of replication of the SIVmac genome in SR cells, while, in contrast, there was no restriction on the replication of the IV-1 genome in these cells. However, because of the lack of functional receptors to SIVmac and HIV-1 at the surface of SR cells, there is specific lentiviral entry.

TRANSFORMASI PLASMID REKOMBINAN pRI_101-AN MEMBAWA SISIPAN GEN cryIII MELALUI Agrobacterium tumefaciens

Sweet potato is a plant that give many benefits because of its high carbohydrate content and is an alternative food source. Behind the benefits of sweet potatoes, there is a threat of pest attack that can cause a decrease in production of up to 19.91%. Sweet potato weevil attack on the leaves, stalks, stems, and tubers. The solution to against this pest attack is by engineering using the cry III gene mediated by A. tumefaciens. The aim of this study was to transform a recombinant plasmid carrying the insertion of the cry III gene into A. tumefaciens cells. The results showed that the recombinant plasmid pRI_101-AN carrying the insertion of the cry III gene was successfully transformed into A. tumefaciens cells by electroporation method by producing 36 recombinant clones on LB media with neomycin and kanamycin antibiotics. Results of colony PCR, DNA bands measuring 1900 bp were successfully amplified using gene-specific primers. Results of the isolation of plasmid DNA bands measuring 12000 bp were obtained from the results of electrophoresis. This size corresponds to the recombinant plasmid pRI_101-AN-cry III.

Generation of a host cell line containing a MAR-rich landing pad for site-specific integration and expression of transgenes.

In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix‐Attachment Region (MAR)‐rich landing pads (LPs), which allow for the simultaneous and site‐specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR‐based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP‐containing host cell lines can be used for the site‐specific integration of several GOIs and thus, generation of transgene‐expressing stable recombinant clones. Transgene expression was shown by site‐specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi‐specific antibody (bsAb). The genetic stability of the herein described LP‐based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR‐rich landing pads can be successfully used for stable expression of one or several GOIs.

The pemphigus monoclonal antibody clone P5G6 (AX353) retains cell-surface staining by immunofluorescence and desmoglein binding by ELISA in an engineered scFv-Fc format

The recombinant human antibody clone P5G6 (AX353) in an engineered scFv-Fc format detects by immunofluorescence cell-surface proteins on cryosections of monkey esophagus. The same antibody detects by ELISA recombinant human desmoglein 3 and desmoglein 1 protein.

Systematic Characterization of bZIP Transcription Factors Required for Development and Aflatoxin Generation by High-Throughput Gene Knockout in Aspergillus flavus.

The basic leucine zipper (bZIP) is an important transcription factor required for fungal development, nutrient utilization, biosynthesis of secondary metabolites, and defense against various stresses. Aspergillus flavus is a major producer of aflatoxin and an opportunistic fungus on a wide range of hosts. However, little is known about the role of most bZIP genes in A. flavus. In this study, we developed a high-throughput gene knockout method based on an Agrobacterium-mediated transformation system. Gene knockout construction by yeast recombinational cloning and screening of the null mutants by double fluorescence provides an efficient way to construct gene-deleted mutants for this multinucleate fungus. We deleted 15 bZIP genes in A. flavus. Twelve of these genes were identified and characterized in this strain for the first time. The phenotypic analysis of these mutants showed that the 15 bZIP genes play a diverse role in mycelial growth (eight genes), conidiation (13 genes), aflatoxin biosynthesis (10 genes), oxidative stress response (11 genes), cell wall stress (five genes), osmotic stress (three genes), acid and alkali stress (four genes), and virulence to kernels (nine genes). Impressively, all 15 genes were involved in the development of sclerotia, and the respective deletion mutants of five of them did not produce sclerotia. Moreover, MetR was involved in this biological process. In addition, HapX and MetR play important roles in the adaptation to excessive iron and sulfur metabolism, respectively. These studies provide comprehensive insights into the role of bZIP transcription factors in this aflatoxigenic fungus of global significance.

Screening of Anticancer and Immunomodulatory Properties of Recombinant pQE-HAS113 Clone Derived from Streptococcus Equi

Objective: Streptococcus equi has been identified to contain the hyaluronan synthase gene (HAS), which could have a role in the cell proliferation activity. The objective of this research was to determine anticancer properties of HAS113 recombinant clone. Methodology: The intracellular and extracellular portions of the expressed gene products of pQE-HAS113 were harvested and evaluate for anticancer properties against prostate cancer cell (PC3) and breast cancer cell (MCF-7). To evaluate anti-proliferative properties of pQE-HAS113, two assays were carried out: MTT assay, which was used for assessing percent cell inhibition and apoptosis assay, through the AO/EB stain of living, apoptotic, as well as necrotic cells. For evaluation of the immunomodulatory properties of pQE-HAS113 on RAW264.7 in terms of IL-6, IFN-γ & IL-8 expression, flow cytometry examination was done. The harvested intracellular and extracellular portions of pQE-HAS113 were tested. Results: The results indicated that the percentage proliferation of both the prostate cancer and breast cancer cells were 2.1% and 7.2% respectively against the intracellular portions of pQE-HAS113. While in case of the extracellular portions of pQE-HAS113 the proliferation rate of both the prostate cancer and breast cancer cells were 6.5% and 6.7% respectively. The PQE-HAS113 intracellular clone gene has shown proinflammatory activity by stimulating the expression of IL-6, INF-γ, & IL-8 in the RAW264.7 macrophage cells. Conclusion: Thus, the results from the present study indicated that the expressed product of recombinant clone of HAS 113, pQE-HAS113 has potential to be used as anti-proliferative and apoptosis inducing substance for prostate and breast cancer. Keywords: Streptococcus equi; breast cancer; cell proliferation; apoptosis ass