BamHI Site Research Articles

Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity.

Effects of human chorionic gonadotropin-producing peripheral blood mononuclear cells on the endometrial receptivity and implantation sites of the mouse uterus.

This research investigated the effects of human chorionic gonadotropin (HCG)-producing peripheral blood mononuclear cells (PBMCs) on the implantation rate and embryo attachment in mice. In this experimental study, a DNA fragment of the HCG gene was cloned into an expression vector, which was transfected into PBMCs. The concentration of the produced HCG was measured using enzyme-linked immunosorbent assay. Embryo attachment was investigated on the co-cultured endometrial cells and PBMCs in vitro. As an in vivo experiment, intrauterine administration of PBMCs was done in plaque-positive female mice. Studied mice were distributed into five groups: control, embryo implantation dysfunction (EID), EID with produced HCG, EID with PBMCs, and EID with HCG-producing PBMCs. Uterine horns were excised to characterize the number of implantation sites and pregnancy rate on day 7.5 post-coitum. During an implantation window, the mRNA expression of genes was evaluated using real-time polymerase chain reaction. DNA fragments were cloned between the BamHI and EcoRI sites in the vector. About 465 pg/mL of HCG was produced in the transfected PBMCs. The attachment rate, pregnancy rate, and the number of implantation sites were substantially higher in the HCG-producing PBMCs group than in the other groups. Significantly elevated expression of the target genes was observed in the EID with HCG-producing PBMCs group. Alterations in gene expression following the intrauterine injection of HCG-producing PBMCs, could be considered a possible cause of increased embryo attachment rate, pregnancy rate, and the number of implantation sites.

Bioinformatics analysis for the purpose of designing a novel multi-epitope DNA vaccine against Leishmania major

Leishmaniasis is one of the main infectious diseases worldwide. In the midst of all the different forms of the disease, Cutaneous Leishmania (CL) has the highest incidence in the world. Many trial vaccines have been developed with the purpose of generating long-term cell-mediated immunity to Leishmania(L) major. As there is not any multi-epitope DNA vaccine with high efficacy against L.major, the aim of this study is to design a new multi-epitope DNA vaccine in order to have effective control upon this infectious disease through the immune bioinformatics. The L.major antigens: Gp63, LACK, TSA, LmSTI1and KMP11 were selected to design a multi-epitope DNA vaccine. The initial structure of the DNA vaccine was designed, benefiting from Gen Bank's website information. Epitopes of MHC-I antigens were predicted through the Immune Epitope Database (IEDB), and the selected epitopes were used to make vaccines construct along with linkers. New multi-epitope vaccine including 459 nucleic acids designed, and inserted between BamH1 and HindIII restriction sites of pCDNA3.1 mammalian expression vector. 12 epitopes among the chosen antigens were selected by two servers (IEDB and ANTIGEN). They had high stability and high antigenic power. Physicochemical features of vaccine measured by ProtParam server, and this structure was thermostable and hydrophilic. it’s a suitable model to study on the animal and human phases. The designed vaccine is expected to be an effective candidate through development of (CL) vaccines. However, the effectiveness of this vaccine should also evaluate in vivo model.

Noncanonical DNA Cleavage by BamHI Endonuclease in Laterally Confined DNA Monolayers Is a Step Function of DNA Density and Sequence.

Cleavage of DNA at noncanonical recognition sequences by restriction endonucleases (star activity) in bulk solution can be promoted by global experimental parameters, including enzyme or substrate concentration, temperature, pH, or buffer composition. To study the effect of nanoscale confinement on the noncanonical behaviour of BamHI, which cleaves a single unique sequence of 6 bp, we used AFM nanografting to generate laterally confined DNA monolayers (LCDM) at different densities, either in the form of small patches, several microns in width, or complete monolayers of thiol-modified DNA on a gold surface. We focused on two 44-bp DNAs, each containing a noncanonical BamHI site differing by 2 bp from the cognate recognition sequence. Topographic AFM imaging was used to monitor end-point reactions by measuring the decrease in the LCDM height with respect to the surrounding reference surface. At low DNA densities, BamHI efficiently cleaves only its cognate sequence while at intermediate DNA densities, noncanonical sequence cleavage occurs, and can be controlled in a stepwise (on/off) fashion by varying the DNA density and restriction site sequence. This study shows that endonuclease action on noncanonical sites in confined nanoarchitectures can be modulated by varying local physical parameters, independent of global chemical parameters.

Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

Abstract. Adibah DI, Fuad AM, Budiarti S, Pratiwi RD. 2022. Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?. Biodiversitas 23: 4289-4296. Kex2 (EC 3.4.21.61) is a serine protease that binds Ca2+ molecules and naturally found intracellularly as a transmembrane protein. Kex2 has a unique function, the conversion process of recombinant protein precursor into mature protein. Kex2 from Saccharomyces cerevisiae has similar functions also with furin in mammals (about 47% of sequence similarity). To gain advantage, extracellular Kex2 would be highly favorable for this process. This study aimed to construct recombinant Kex2 that could be produced extracellularly in Pichia pastoris host through pD902-KEX2-699 vector (synthetic) with FLAG-tag and 6 His-tag by removing most of C-terminal region, including transmembrane domain (TMD) from KEX2 gene sequence. Constructed KEX2 is the KEX2-650 variant with TMD deletion and cytoplasmic domain. The recombinant plasmid was constructed through site-directed mutagenesis using FP-Kex2-699 and RP-Kex2-650 primers, including the BamHI site for plasmid religation. PCR site-directed mutagenesis produces an amplicon DNA with an expected length of 5551 bp. After restriction (BamHI) and religation, the plasmid was reintroduced into Escherichia coli DH5? and obtained 16 colonies. Verification PCR target gene showed that clones number 9 produced an amplicon of expected length (646 bp). DNA sequencing analysis confirmed that TMD was removed from the gene construct to form the KEX2-650 construct.

Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain.

Strand-specific DNA nicking endonucleases (NEases) typically nick 3–7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction. The mutant was fused to a 76-amino acid (aa) DNA nicking domain of phage Gamma HNH (gHNH) NEase. The chimeric enzyme was purified, and it was shown to nick downstream of a composite site 5′ GGATCC-N(4-6)-AC↑CGR 3′ (R, A, or G) or to nick both sides of BamHI site at the composite site 5′ CCG↓GT-N5-GGATCC-N5-AC↑CGG 3′ (the down arrow ↓ indicates the strand shown is nicked; the up arrow↑indicates the bottom strand is nicked). Due to the attenuated activity of the small nicking domain, the fusion nickase is active in the presence of Mn2+ or Ni2+, and it has low activity in Mg2+ buffer. This work provided a proof-of-concept experiment in which a chimeric NEase could be engineered utilizing the binding specificity of a Type II restriction endonucleases (REases) in fusion with a nicking domain to generate infrequent nickase, which bridges the gap between natural REases and homing endonucleases. The engineered chimeric NEase provided a framework for further optimization in molecular diagnostic applications.

Genomics-Enabled Novel Insight Into the Pathovar-Specific Population Structure of the Bacterial Leaf Streak Pathogen Xanthomonas translucens in Small Grain Cereals.

The Gram-negative bacterium Xanthomonas translucens infects a wide range of gramineous plants with a notable impact on small grain cereals. However, genomics-informed intra-species population structure and virulence repertories of the pathogen have rarely been investigated. In this study, the complete genome sequences of seven X. translucens strains representing an entire set of genetic diversity of two pathovars X. translucens pv. undulosa and X. translucens pv. translucens is provided and compared with those of seven publicly available complete genomes of the pathogen. Organization of the 25 type III secretion system genes in all the 14 X. translucens strains was exactly the same, while TAL effector genes localized singly or in clusters across four loci in X. translucens pv. translucens and five to six loci in X. translucens pv. undulosa. Beside two previously unreported endogenous plasmids in X. translucens pv. undulosa, and variations in repeat variable diresidue (RVD) of the 14 strains, tal1a of X. translucens pv. translucens strain XtKm8 encode the new RVDs HE and YI which have not previously been reported in xanthomonads. Further, a number of truncated tal genes were predicted among the 14 genomes lacking conserved BamHI site at N-terminus and SphI site at C-terminus. Our data have doubled the number of complete genomes of X. translucens clarifying the population structure and genomics of the pathogen to pave the way in the small grain cereals industry for disease resistance breeding in the 21st century’s agriculture.

Prenatal Diagnosis of β-Thalassemias by Amniocentesis: Linkage Analysis Using Multiple Polymorphic Restriction Endonuclease Sites

In order to assess the applicability of multiple restriction endonuclease analyses of amniocyte DNA to the prenatal diagnosis of beta-thalassemias in general, we studied 12 consecutive couples at risk. DNA of both members of the 12 couples and a previous offspring of each was analyzed for the presence of 4 polymorphic restriction endonuclease sites: the Hpa I site 3' to the beta-globin gene, the Hind III site in the G gamma gene, the Hind III site in the A gamma gene, and the Bam HI site 3' to the beta-gene. Linkage disequilibrium between these sites and beta A or beta thal genes was not found, presumably due to the heterogeneity of beta thal genes. However, the high frequency of polymorphism at these sites allowed differentiation of beta A-bearing chromosomes from beta thal or beta S-bearing chromosomes in both members of 6 couples. In these couples, complete prenatal diagnosis by linkage analysis of amniocyte DNA would be possible. In the remaining 6 couples, beta A and beta thal chromosomes could be discriminated in one member. In about 50% of the pregnancies of these couples, exclusion of beta-thalassemia is possible by this analysis. These data suggest that when linkage analysis of polymorphic restriction endonuclease sites is carried out, prenatal diagnosis of beta-thalassemia states can be accomplished by amniocentesis alone in 75% of pregnancies at risk.

Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in E. Coli for Cancer Immunotherapy

: Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe E. coli expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for ex vivo stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells. A bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the HindIII and BamHI site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the E. coli (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole. Analyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein. Results showed efficient production of the bsAb in E. coli for future large-scale purification.

Tạo dòng và biểu hiện interleukin 2 (IL-2) người tái tổ hợp trong Escherichia coli

Immunotherapy using interleukin 2 (IL-2) was approved by FDA for the treatment of some solid tumors and for the combination with AIDS/HIV’s HAART. In order to produce recombinant interleukin 2 with low price, the project was initiated to clone and express IL-2 in Escherichia coli. The il2 gene was amplified by PCR, using the pIDT-IL2 as template with two specific primers (IL2-F and IL2-R) containing BamHI and NdeI sites. The PCR product was digested with BamHI and NdeI then inserted into an expression vector pET-His to construct a recombinant plasmid termed pET-il2 capable of expressing IL-2 under the control of T7 promoter. The recombinant plasmid pET-il2 was transformed into E. coli strain Origami(DE3) to form the Origami(DE3)/pET-il2 clone. This clone was cultured and induced with 0.5 mM IPTG over-expressed IL-2 protein as inclusion body in cytoplasm. The expressing of the target protein was confirmed by SDS-PAGE and Western Blot with anti-IL2 antibody.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Genomic characterization of two Orf virus isolates from Jilin province in China.

Orf virus (ORFV), a typical member of the Parapoxvirus genus within the family Poxviridae, which is the causative agent of Orf, a common epitheliotropic viral disease of sheep, goats, wild ruminants, and humans. In the present study, we sequenced the complete genomic sequences of two ORFV strains (ORFV-SY17, isolated from sheep, and ORFV-NA17, isolated from goat) and conducted the comparative analysis of multiple ORFVs. The complete genomic sequence of ORFV-SY17 was at length of 140,413bp, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 4267bp at both ends. The ORFV-NA17 strain displayed the similar genome structure with ORFV-SY17. The whole genomic sequence of ORFV-NA17 strain was 139,287bp in length and contained 132 ORFs flanked by ITRs of 3974bp. The overall G+C contents of ORFV-SY17 and ORFV-NA17 genome sequences were about 63.8% and 63.7%, respectively. The ITR sequences analysis showed that ORFV-SY17 and ORFV-NA17 contained the terminal BamHI sites and conserved telomere resolution sequences at both ends of their genome. In addition, comparative analysis of ORFs among ORFV-SY17, ORFV-NA17, and other ORFV strains revealed several sequence variations caused by insertions or deletions, especially in ORFs 005 and 116, which were very likely associated with host species. Phylogenetic analysis based on the complete genome sequences revealed that ORFV-SY17 was genetically closely related to NA1/11 and HN3/12 strains derived from sheep, while ORFV-NA17 was closely related to YX strain derived from goat. The multiple alignment of deduced amino acid sequences further revealed the genetic relationship between host species and genetic variations of ORFV strains. Taken together, the availability of genomic sequences of ORFV-SY17 and ORFV-NA17 strains from Jilin Province will aid in our understanding of the genetic diversity and evolution of ORFV strains in this region and can assist in distinguishing between ORFV strains that originate in sheep and goats.

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays.

ObjectiveMutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a.MethodsBioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR.ResultsGel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = −0.656, p<0.05).ConclusionLDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

Specific cytotoxicity of epidermal groth factor variantIII oriented chimeric antigen receptor-engineered T cells on SMMC-7721

Objective To observe the specific killing effect of anti-epidermal groth factor variantⅢ (EGFRvⅢ) chimeric antigen receptor modified T cells (EGFRvⅢ/2CAR-T) on liver cancer cell SMMC-7721. Methods Expression cassette of EGFRvⅢ/2CAR (1 374 bp) was inserted into XbaⅠ and BamHⅠ sites of piggybac transposon vector PB513B-1 (PB-EGFRvⅢ/2CAR), the latter together with transposase plasmid was then transducted into human peripheral blood T cells by electroporation (1×20 ms, 840 V). The expression of EGFRvⅢ/2CAR on T cells was detected by flow cytometry. The expression of EGFRvⅢ in SMMC-7721 was detected by Western blotting. After EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721, the killing effect was detected by lactate dehydrogenase (LDH) release test and cytokine interferon (IFN)-γ secretion. Results The recombinant vector PB-EGFRvⅢ/2CAR was confirmed by XbaⅠ and BamHⅠ enzyme digestion, electrophoresis and DNA sequencing analysis. The expression of EGFRvⅢ/2CAR on T cells was detected by Flow cytometry (55.5%). Western blotting analysis showed that a protein band of approximate 145 000 molecular weight was observed in SMMC-7721 cells, which corresponds to the calculated molecular weight of EGFRvⅢ. EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721 for 24 h, LDH release test showed that tumor specific killing was positively correlated with E∶T ratio (E∶T=1∶1, 5∶1, 10∶1, 15∶1, 20∶1); enzyme linked immunosorbent assay (ELISA) showed that cytokine IFN-γ secretion was (1 060.1±89.0) pg/ml, which was significantly higher than control group (P=0.000). Conclusion Anti-EGFRvⅢ CAR-T cells can kill specifically EGFRvⅢ+ liver cancer cell SMMC-7721, providing research basis for immunotherapy of hepatic cancer. Key words: Epidermal groth factor variantⅢ; Chimeric antigen receptor; Immunotherapy; PiggyBac transposon

Molecular identification of imported red wood ant populations in the Campigna Biogenetic Nature Reserve (Foreste Casentinesi National Park, Italy)

During the second half of the XIX century, several species of red wood ants (Formica rufa group) were used as biological control agents and introduced to the Italian Apennine mountains where they were formerly absent. Among these, Formica lugubris was the most widely introduced alien species. In 1996, however, a taxonomic revision defined two sibling species, F. lugubris and F. paralugubris, each with distinct ecological and behavioural characteristics that differently affect their spatial distribution. The first one can be both monodomous and polydomous with a low number of interlinked nests, and it is obliged to mate by nuptial flight. Conversely, F. paralugubris is usually unicolonial, with a considerable portion of intranest mates. Discrimination between the two species using morphological characters is possible but requires considerable taxonomic expertise. Restriction analysis, whereby presence/absence of a BamHI site in the mitochondrial DNA COI gene results in different restriction profiles, provides an alternative efficient method to unambiguously distinguish F. lugubris from F. paralugubris. We applied this method to identify ant species introduced to several locations in the Campigna Forest of the Foreste Casentinesi, Monte Falterona and Campigna National Park, one of the Italian locations mostly affected by alien ant introductions. This area has considerable ecological relevance. Given the potential impact of ants on native arthropod communities, it is of the most importance to identify which species has been introduced. Contrary to previous assumptions, results of our analysis clearly show that only F. paralugubris is present in the National park.

Isolation of equine herpesvirus 3 (EHV-3) from equine coital exanthema of two stallions and sero-epidemiology of EHV-3 infection in Japan

In the spring of 2015, two stallions reared in Farms A and B in Hokkaido in Japan showed symptoms of equine coital exanthema. Equine herpesvirus 3 (EHV-3) was isolated from penis swab samples of both stallions, and the isolates from each stallion in Farms A and B were designated as SS-1 and YS-1 strains, respectively. BamHI restriction profiles of SS-1 and Japanese reference strain Iwate-1 were indistinguishable, but the BamHI-A fragment of YS-1 was larger than those of SS-1 and Iwate-1 by 1.9 kbp because of the lack of two BamHI sites. Nucleotide sequence analyses of glycoprotein G (gG), gB, gC and VP13/14 coding regions revealed that SS-1 and YS-1 had 99.77% to 100% identities to each other. These results suggested that the origins of SS-1 and YS-1 were different. For a sero-epidemiological survey, serum neutralizing tests using SS-1 against 319 sera of horses from eight farms in Hokkaido were conducted. Six of the eight farms were EHV-3 antibody-positive, and positive rates ranged from 2.6% to 17.6%. To determine the infection time of four EHV-3 antibody-positive horses, a retrospective study was conducted. Infection time of the four horses was in the breeding season, and re-infection or reactivation of latently infected EHV-3 might have occurred in one horse. However, these four horses had never shown any clinical symptoms. The results suggested that several EHV-3 strains are distributed in Japan and that infection is maintained widely in horses without clinical symptoms.

Expression of Recombinant Sugarcane Streak Mosaic Virus Coat Protein Gene in Escherichia coli

Sugarcane streak mosaic virus (SCSMV) is an important virus causing mosaic disease in sugarcane and transmitted through the cutting cane. Commercial antiserum to detect SCSMV and to monitor the disease development is not available. The research was conducted to produce antigen of SCSMV coat protein (SCSMV-CP) through overexpressing it on bacterial expression which will be used for antiserum production. SCSMV-CP was amplified using specific primers for CP gene containing BamHI and HindIII restriction enzyme sites and cloned into pTZ57R/T. Subsequently, the SCSMV-CP was subcloned into pET28a and transformed on Escherichia coli BL21(DE3) and Rosetta-gami(DE3)pLysS. The concentration of isopropyl β-d-thiogalactopyranoside (IPTG), incubation temperature, and bacterial harvesting time after IPTG induction were optimized. SCSMV-CP gene was successfully amplified with size ±855 bp, subcloned into vector expression, and expressed in insoluble fraction either in both bacterial host. Optimal protein expression of SCSMV-CP recombinant was obtained at 25°C with IPTG concentration 0.25–1.00 mM and harvested at 9–12 hours after IPTG induction in E. coli BL21(DE3), and at 30°C with IPTG concentration 0.25–1.00 mM and harvested 3–12 hours after IPTG induction in E. coli Rosetta(DE3)pLysS. SDS-PAGE analysis showed that protein size of SCSMV-CP recombinant was ±35.4 kDa.

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg(2+) ion in a manner that allows it to assist in transition state stabilization. The Mg(2+) ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg(2+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg(2+). Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS.

Construction of a recombinant baculovirus expressing the hemaglutimin (ha) of an A/H5N1 clade 2.3.3.1b virus

Highly pathogenic avian influenza A (H5N1) virus is still a threat to public health, especially, the emergence of a new clade recently with more virulent and resistant to the vaccine developed from the previously circulating clade has raised the need for development of new vaccines. With the ultimate aim of producing influenza virus-like particles (VLPs) vaccine, in this study we created recombinant baculovirus expressing Hemagglutini (HA) from H5N1 virus clade 2.3.2.1b isolated in Quang Ngai province (DkQN37/11) – which is resistant to NIBRG14 (clade 1) currently used in Vietnam. The HA - encoding gene was amplified by PCR with a specific pair of primers, which has Kozak sequence, initation codon and BamHI site at the 5’ of the forward primer and the recognition sequence of HindIII at the 5' of the reverse primer. The PCR product was cloned into pCR2.1 and sequenced then inserted into the baculovirus vector (pBluBac4.5 / V5-His-TOPO, Invitrogen) at BamHI and HindIII sites to yield pBuBacHA. Vector pBluBacHA then co-transfected with baculovirus DNA into insect cells (Sf9) to generate recombinant baculovirus. The presence of ha gene and the expression of recombinant HA protein in the recombinant virus were detemined by PCR and Western blot using anti-HA antibody. PCR and Western blot results confirmed that we have successfully created a recombinant baculovirus expressing surface antigens of influenza A/H5N1 clade 2.3.2.1b. Our work provides essential material for further study to develop A/H5N1 VLP vaccine in Vietnam.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.