Journal of Microbiology

Review

Adenoviral Vector System: A Comprehensive Overview of Constructions, Therapeutic Applications and Host Responses: Anyeseu Park, Jeong Yoon Lee; J. Microbiol. 2024;62(7):491-509. Published online July 22, 2024; DOI: https://doi.org/10.1007/s12275-024-00159-4

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Adenoviral vectors are crucial for gene therapy and vaccine development, offering a platform for gene delivery into host cells. Since the discovery of adenoviruses, first-generation vectors with limited capacity have evolved to third-generation vectors flacking viral coding sequences, balancing safety and gene-carrying capacity. The applications of adenoviral vectors for gene therapy and anti-viral treatments have expanded through the use of in vitro ligation and homologous recombination, along with gene editing advancements such as CRISPR-Cas9. Current research aims to maintain the efficacy and safety of adenoviral vectors by addressing challenges such as pre-existing immunity against adenoviral vectors and developing new adenoviral vectors from rare adenovirus types and non-human species. In summary, adenoviral vectors have great potential in gene therapy and vaccine development. Through continuous research and technological advancements, these vectors are expected to lead to the development of safer and more effective treatments.

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Engineering an oncolytic adenoviral platform for precise delivery of antisense peptide nucleic acid to modulate PD-L1 overexpression in cancer cells
Andrea Patrizia Falanga, Francesca Greco, Monica Terracciano, Stefano D’Errico, Maria Marzano, Sara Feola, Valentina Sepe, Flavia Fontana, Ilaria Piccialli, Vincenzo Cerullo, Hélder A. Santos, Nicola Borbone
International Journal of Pharmaceutics.2025; 668: 124941. CrossRef
Enhancing precision in cancer treatment: the role of gene therapy and immune modulation in oncology
Emile Youssef, Brandon Fletcher, Dannelle Palmer
Frontiers in Medicine.2025;[Epub] CrossRef
Protein-Based Degraders: From Chemical Biology Tools to Neo-Therapeutics
Lisha Ou, Mekedlawit T. Setegne, Jeandele Elliot, Fangfang Shen, Laura M. K. Dassama
Chemical Reviews.2025; 125(4): 2120. CrossRef
Intestinal mucus: the unsung hero in the battle against viral gastroenteritis
Waqar Saleem, Ateeqa Aslam, Mehlayl Tariq, Hans Nauwynck
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Chromatin structure and gene transcription of recombinant p53 adenovirus vector within host
Duo Ning, Yuqing Deng, Simon Zhongyuan Tian
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Molecular Engineering of Virus Tropism
Bo He, Belinda Wilson, Shih-Heng Chen, Kedar Sharma, Erica Scappini, Molly Cook, Robert Petrovich, Negin P. Martin
International Journal of Molecular Sciences.2024; 25(20): 11094. CrossRef
Antisolvent 3D Printing of Gene-Activated Scaffolds for Bone Regeneration
Andrey Vyacheslavovich Vasilyev, Irina Alekseevna Nedorubova, Viktoria Olegovna Chernomyrdina, Anastasiia Yurevna Meglei, Viktoriia Pavlovna Basina, Anton Vladimirovich Mironov, Valeriya Sergeevna Kuznetsova, Victoria Alexandrovna Sinelnikova, Olga Anatol
International Journal of Molecular Sciences.2024; 25(24): 13300. CrossRef

Journal Articles

Genetically Engineered CLDN18.2 CAR-T Cells Expressing Synthetic PD1/CD28 Fusion Receptors Produced Using a Lentiviral Vector: Heon Ju Lee, Seo Jin Hwang, Eun Hee Jeong, Mi Hee Chang; J. Microbiol. 2024;62(7):555-568. Published online May 3, 2024; DOI: https://doi.org/10.1007/s12275-024-00133-0

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Abstract: This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.

Vagococcus zengguangii sp. nov., isolated from yak faeces: Yajun Ge , Dong Jin , Xin-He Lai , Jing Yang , Shan Lu , Ying Huang , Han Zheng , Xiaoyan Zhang , Jianguo Xu; J. Microbiol. 2021;59(1):1-9. Published online December 23, 2020; DOI: https://doi.org/10.1007/s12275-021-0406-3

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Abstract

Two unknown Gram-stain-positive, catalase- and oxidasenegative, non-motile, and coccus-shaped bacteria, designated MN-17T and MN-09, were isolated from yaks faeces (Bos grunniens) in the Qinghai-Tibet Plateau of China. 16S rRNA gene sequence-based comparative analyses revealed that the two strains were grouped within the genus Vagococcus, displaying the highest similarity with Vagococcus xieshaowenii CGMCC 1.16436T (98.6%) and Vagococcus elongatus CCUG 51432T (96.4%). Both strains grew optimally at 37°C and pH 7.0 in the presence of 0.5% (w/v) NaCl. The complete genome of MN-17T comprises 2,085 putative genes with a total of 2,190,262 bp and an average G + C content of 36.7 mol%. The major fatty acids were C16:0 (31.2%), C14:0 (28.5%), and C18:1ω9c (13.0%); the predominant respiratory quinone was MK-7 (68.8%); the peptidoglycan type was A4α(L-Lys-DAsp); and the major polar lipid was diphosphatidylglycerol. Together, these supported the affiliation of strain MN-17T to the genus Vagococcus. In silico DNA-DNA hybridization and the average nucleotide identity values between MN-17T and all recognized species in the genus were 21.6–26.1% and 70.7–83.0%, respectively. MN-17T produced acid from D-cellobiose, D-fructose, glycerol, D-glucose, N-acetyl-glucosamine, gentiobiose, D-mannose, D-maltose, D-ribose, Dsaccharose, salicin, D-trehalose, and D-xylose. These results distinguished MN-17T and MN-09 from closely related species in Vagococcus. Thus, we propose that strains MN-17T and MN-09 represent a novel species in the genus Vagococcus, with the name Vagococcus zengguangii sp. The type strain is MN-17T (= CGMCC 1.16726T = GDMCC 1.1589T = JCM 33478T).

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Vagococcus proximus sp. nov. and Vagococcus intermedius sp. nov., originating from modified atmosphere packaged broiler meat
Per Johansson, Elina Jääskeläinen, Elina Säde, Johanna Björkroth
International Journal of Systematic and Evolutionary Microbiology .2023;[Epub] CrossRef
Phenotypic and genomic characteristics of Brevibacterium zhoupengii sp. nov., a novel halotolerant actinomycete isolated from bat feces
Yuyuan Huang, Lingzhi Dong, Jian Gong, Jing Yang, Shan Lu, Xin-He Lai, Dong Jin, Qianni Huang, Ji Pu, Liyun Liu, Jianguo Xu
Journal of Microbiology.2022; 60(10): 977. CrossRef

Research Support, Non-U.S. Gov'ts

Identification of Porcine Endogenous Retrovirus (PERV) packaging sequence and development of PERV packaging viral vector system: Jiwon Choi , Hoon-mi Kim , Jong Kwang Yoon , Yeondong Cho , Hee-Jung Lee , Kang Chang Kim , Chang-Kyu Kim , Gye-Woong Kim , Young Bong Kim; J. Microbiol. 2015;53(5):348-353. Published online May 3, 2015; DOI: https://doi.org/10.1007/s12275-015-5134-0

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Abstract

Studies of the retroviruses have focused on the specific interaction of the nucleocapsid protein with a packaging signal in the viral RNA as important for this selectivity, but the packaging signal in porcine endogenous retrovirus (PERV) has not been defined. Herein, we identified and analyzed this packaging signal in PERV and found hairpin structures with conserved tetranucleotides in their loops and nucleocapsid recognition sequences; both of which are key elements in the viral packaging signal of MLV. We evaluated packaging efficiency of sequence variants isolated from viral and proviral integrated genomes. All viral packaging sequences (Ψ) were identical, while five distinct packaging sequences were identified from proviral sources. One proviral sequence (Ψ1) was identical to that of the viral Ψ and had the highest packaging efficiency. Three variants (Ψ2, Ψ3, Ψ4) maintained key elements of the viral packaging signal, but had nucleotide replacements and consequently demonstrated reduced packaging efficiency. Despite of the same overall hairpin structure, the proviral variant (Ψ5) had only one GACG sequence in the hairpin loop and showed the lowest packaging efficiency other than ΔΨ, in which the essential packaging sequence was removed. This result, thus, defined the packaging sequences in PERV and emphasized the importance of nucleotide sequence and RNA structure in the determination of packaging efficiency. In addition, we demonstrate efficient infection and gene expression from the PERVbased viral vector, which may serve as a novel alternative to current retroviral expression systems.

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Porcine Endogenous Retrovirus (PERV) – Molecular Structure and Replication Strategy in the Context of Retroviral Infection Risk of Human Cells
Krzysztof Łopata, Emilia Wojdas, Roman Nowak, Paweł Łopata, Urszula Mazurek
Frontiers in Microbiology.2018;[Epub] CrossRef

An Easy Way for the Rapid Purification of Recombinant Proteins from Helicobacter pylori Using a Newly Designed Expression Vector: Hyung-Lyun Kang , Jin-Sung Jo , Soon-Uck Kwon , Jae-Young Song , Ji-Hyun Seo , Myung-Je Cho , Seung-Chul Baik , Hee-Shang Youn , Kwang-Ho Rhee , Woo-Kon Lee; J. Microbiol. 2014;52(7):604-608. Published online June 28, 2014; DOI: https://doi.org/10.1007/s12275-014-3679-y

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Abstract

We constructed a H. pylori expression vector which consisted of both a His-tag and a GST tag as purification tools for recombinant protein and a chloramphenicol resistant cat gene as a reporter. The backbone of the vector pBK contained an ColEI origin of replication and a kanamycin resistant gene. A set of oligos for the His-tag and the PCR product of gst (glutathione S-transferase) gene were inserted sequentially in frame in the multi-cloning site of pBK. The orf of cat was inserted downstream of the gst to generate pBKHGC. The 3' part of H. pylori clpB and flaA were cloned into the vector which was introduced into H. pylori. Recombinant proteins were purified by GSH affinity column, digested with thrombin and were analyzed by western blotting. The final recombinant proteins were successfully purified.

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Citations to this article as recorded by

Gene polymorphisms of pathogenic Helicobacter pylori in patients with different types of gastrointestinal diseases
Yu-Li Chen, Xiao-Qiang Mo, Gan-Rong Huang, Yan-Qiang Huang, Juan Xiao, Li-Juan Zhao, Hong-Yu Wei, Qian Liang
World Journal of Gastroenterology.2016; 22(44): 9718. CrossRef

Novel Bifidobacterium Promoters Selected Through Microarray Analysis Lead to Constitutive High-Level Gene Expression: Yan Wang , Jin Yong Kim , Myeong Soo Park , Geun Eog Ji; J. Microbiol. 2012;50(4):638-643. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-1591-x

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Abstract: For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.

Development of a Suicidal Vector-Cloning System Based on Butanal Susceptibility Due to an Expression of YqhD Aldehyde Reductase: Changhan Lee , Chankyu Park; J. Microbiol. 2012;50(2):249-255. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1438-5

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Abstract: Previously, we observed butanal/propanal sensitivity of Escherichia coli K-12 when cells overexpress YqhD protein, a NADPH dependent aldehyde reductase, possibly due to an accumulation of butanol/propanol in vivo as the reaction products. Based on this finding, we developed a suicidal vector-cloning system derived from pUC19, in which lacZ was substituted with the yqhD gene. As a result, when foreign DNA was inserted into its multiple cloning sites by disrupting an expression of YqhD, the recombinants survived on butanal/propanal containing plate, whereas cells containing the YqhD vector died because of the alcohol production by YqhD. The cloning efficiency, estimated based on colony PCR and enzyme digestion, was achieved more than 90% when the suicidal vector system was used. Moreover, the plasmid vector itself was stably maintained in the cell, presumably due to its ability to remove toxic aldehydes being accumulated in E. coli cell by metabolic stress.

Validation Study

Generation of Expression Vectors for High-Throughput Functional Analysis of Target Genes in Schizosaccharomyces pombe: Jiwon Ahn , Chung-Hae Choi , Chang-Mo Kang , Chun-Ho Kim , Hee-Moon Park , Kyung-Bin Song , Kwang-Lae Hoe , Misun Won , Kyung-Sook Chung; J. Microbiol. 2009;47(6):789-795. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0010-4

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Abstract: An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, ura4+ and Saccharomyces cerevisiae LEU2 complementing leu1. These vectors contain 3 different strengths of the inducible promoter nmt1, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.

Research Support, Non-U.S. Gov'ts

Construction of an Escherichia-Pseudomonas Shuttle Vector Containing an Aminoglycoside Phosphotransferase Gene and a lacZ'''' Gene for α-Complementation: Bheong-Uk Lee , Ja-Heon Hong , Hyung-Yeel Kahng , Kye-Heon Oh; J. Microbiol. 2006;44(6):671-673.; DOI: https://doi.org/2458 [pii]

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Abstract: A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminoglycoside phosphotransferase gene (aph) from Tn903, a lacZ'''' gene for α-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DH5α and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

Cloning and Sequence Analysis of a Glyceraldehyde-3-phosphate Dehydrogenase Gene from Ganoderma lucidum: Xu Fei , Ming Wen Zhao , Yu Xiang Li; J. Microbiol. 2006;44(5):515-522.; DOI: https://doi.org/2446 [pii]

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Abstract: A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

Expression of human hypoxanthine phosphoribosyltransferase in Insect Cells Using a Baculovirus Vector: Lee, Chong Ho , Yang, Ji Won , Baek, Sang Ki , Yang, jai Myung; J. Microbiol. 1995;33(1):85-90.

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Abstract: The hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to the mononucleotide, IMP and GMP, respectively. For construction of recombinant AcNPV carrying human HPRT, a transfer vector p918 constructed by cloning full-length cDNA for human HPRT into pVL1393 and AnNPV genomic DNA were co-transfected into Sf21 cells. The tissue culture fluid containing extracellular virus was plaque assayed and a recombinant virus with occlusion minus phenotype was obtained by three rounds of plaque purification. Southern blot analysis and PRC results confirmed the insertion of the human HPRT cDNA within the recombinant virus(AcHPRT918). SDS-PAGE and Western blot analysis of the Sf21 cell extracts infected with AcHPRT918 indicated that human HPRT was expressed in insect cell. Large quantities of functional HPRT expressed in insect cells would facilitate characterization of the biological properties of this enzyme.

Construction of secretion vectors using the α-amylase signal sequence of bacillus subtilis NA64: Kim, Sung Il , Lee, Se Yong; J. Microbiol. 1996;34(1):74-81.

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Abstract: Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the α-amylase gene from an α-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the α-amylase gene for easy replacement of various foreign structural genes. To evaluate this secretion vectors, the β-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active β-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each β-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed β- lactamases were located idn the culture medium. The amount of the secreted β-lactamase was about 80% of the total secreted proteins in the culture medium.

A plasmid vector faciliting gene expression in both yeast and mammalian cells: Lee , Tae Ho; J. Microbiol. 1997;35(2):149-151.

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Abstract: A plasmid vector with combined features of yeast shuttle vector and mammalian expression vector was constructed to facilitate expression of cloned gene in both cell-types. All necessary elements required for plasmid maintenance and selection in E. coli, yeast and mammalian cells were size-economically arranged in this plasmid. The human cytomegalovirus (CMV) immediate early promoter and yeast GAL1 promoter were sequentially placed in front of the gene to be expressed. The synthetic splicing donor and acceptor sequences were inserted into the immediate upstream and downstream of the GAL1 promotor, allowing the CMV promotor to direct the expression of a given gene in mammalian cell environment by splicing out the interfering GAL1 promotor sequence. When the resulting vector containing LacZ as a gene was introduced into yeast and mammalian cells, both cells efficiently produced β-galactosidase, dimonstrating its dual host usage.

Construction of a Hexapeptide Library using Phage Display for Bio-panning: Cho, Won Hee , Yoo, Seung Ku; J. Microbiol. 1999;37(2):97-101.

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Abstract: Random hexapeptide library on the surface of filamentous bacteriophage was constructed using the SurfZAP vector. The size of the library was approximately 105. The peptide insert was flanked by two cysteines to constrain the peptide structure with a disulfide bond. This library was screened for the topoisomerase II binding peptide. Dramatic enrichment of the fusion phage over the VCS M13 helper phage was demonstrated by bio-panning affinity selection.

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