-
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Jun 2024To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of , and explore its preliminary applications.
OBJECTIVE
To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of , and explore its preliminary applications.
METHODS
The GRA24 coding sequences of different strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA).
RESULTS
SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following invasion in host cells.
CONCLUSIONS
The polyclonal antibody against the GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.
Topics: Animals; Toxoplasma; Protozoan Proteins; Mice, Inbred BALB C; Mice; Antibodies, Protozoan; Female; Recombinant Proteins; Antibody Specificity; Antigens, Protozoan
PubMed: 38952314
DOI: 10.16250/j.32.1374.2024083 -
Methods in Molecular Biology (Clifton,... 2024Mammalian cell lines are one of the best options when it comes to the production of complex proteins requiring specific glycosylation patterns. Plasmid DNA transfection...
Mammalian cell lines are one of the best options when it comes to the production of complex proteins requiring specific glycosylation patterns. Plasmid DNA transfection and stable cell lines are frequently used for recombinant protein production, but they are expensive at large scale or can become time-consuming, respectively. The BacMam baculovirus (BV) is a safe and cost-effective platform to produce recombinant proteins in mammalian cells. The process of generating BacMam BVs is straightforward and similar to the generation of "insect" BVs, with different commercially available platforms. Although there are several protocols that describe recombinant protein expression with the BacMam BV in adherent cell lines, limited information is available on suspension cells. Therefore, it is of relevance to define the conditions to produce recombinant proteins in suspension cell cultures with BacMam BVs that facilitate bioprocess transfer to larger volumes. Here, we describe a method to generate a high titer BacMam BV stock and produce recombinant proteins in suspension HEK293 cells.
Topics: Baculoviridae; Humans; Recombinant Proteins; HEK293 Cells; Animals; Transfection; Genetic Vectors; Cell Culture Techniques; Gene Expression; Glycosylation
PubMed: 38951347
DOI: 10.1007/978-1-0716-3961-0_25 -
Methods in Molecular Biology (Clifton,... 2024Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free...
Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free methods for protein expression require fewer steps for obtaining protein expression by eliminating virus amplification and measuring the infectivity of the virus. The nonviral method uses a nonlytic plasmid to transfect the gene of interest into the insect cells instead of using baculovirus, a lytic system. In this chapter, we describe one of the transfection methods, which uses polyethyleneimine (PEI) as a DNA delivery material into the insect cells to express the recombinant protein in both adherent and suspension cells.
Topics: Animals; Recombinant Proteins; Transfection; Polyethyleneimine; Plasmids; Insecta; Sf9 Cells; Cell Line; Gene Expression; Spodoptera
PubMed: 38951345
DOI: 10.1007/978-1-0716-3961-0_23 -
Methods in Molecular Biology (Clifton,... 2024This chapter outlines the use of TOPO cloning for streamlined generation of a recombinant plasmid containing your gene of interest for use in the Bac-to-Bac Baculovirus...
This chapter outlines the use of TOPO cloning for streamlined generation of a recombinant plasmid containing your gene of interest for use in the Bac-to-Bac Baculovirus Expression System.
Topics: Plasmids; Cloning, Molecular; Genetic Vectors; Baculoviridae; Chromosomes, Artificial, Bacterial
PubMed: 38951327
DOI: 10.1007/978-1-0716-3961-0_5 -
Archives of Microbiology Jun 2024A Gram-stain-negative, aerobic, rod-shaped and motile strain HL-JVS1, was isolated from the gastric tract of a juvenile Pacific white shrimp. Molecular phylogenetic...
A Gram-stain-negative, aerobic, rod-shaped and motile strain HL-JVS1, was isolated from the gastric tract of a juvenile Pacific white shrimp. Molecular phylogenetic analysis based on 16S rRNA gene sequences of strain HL-JVS1 revealed its affiliation with the genus Pleionea, with close relatives including Pleionea mediterranea MOLA115 (97.5%) and Pleionea sediminis S1-5-21 (96.2%). The complete genome of strain HL-JVS1 consisted of a circular 4.4 Mb chromosome and two circular plasmids (6.6 and 35.0 kb) with a G + C content of 43.1%. The average nucleotide identity and digital DNA-DNA hybridization values between strain HL-JVS1 and the type strains of described Pleionea species were 69.7-70.4% and 18.3-18.6%, respectively. Strain HL-JVS1 grew at 10-40 °C (optimum, 30 °C) in the presence of 0.5 - 9.0% (w/v) sea salts (optimum, 2.0 - 2.5%), and at pH range of 5.5 - 10.0 (optimum, pH 6.5). The major fatty acids (> 10%) were summed feature 9 (iso-C ω9c and/or C 10-methyl) (23.3%), iso-C (14.5%), iso-C 3-OH (13.8%) and iso-C (11.0%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, two unidentified aminolipids, and two unidentified lipids. The respiratory quinone was ubiquinone-8. The comprehensive phylogenetic, phylogenomic, phenotypic and chemotaxonomic results showed that strain HL-JVS1 is distinct from other Pleionea species. Hence, we propose strain HL-JVS1 as a novel species belonging to the genus Pleionea, for which the name Pleionea litopenaei sp. nov. is proposed with HL-JVS1 (= KCCM 90514 = JCM 36490) as the type strain.
Topics: Animals; Penaeidae; Phylogeny; Base Composition; RNA, Ribosomal, 16S; Fatty Acids; DNA, Bacterial; Bacterial Typing Techniques; Nucleic Acid Hybridization; Sequence Analysis, DNA; Genome, Bacterial; Planococcaceae; Gastrointestinal Tract; Phospholipids
PubMed: 38951206
DOI: 10.1007/s00203-024-04064-7 -
Applied Microbiology and Biotechnology Jun 2024Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and...
Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.
Topics: Escherichia coli; Cloning, Molecular; Plasmids; Recombination, Genetic; Polymerase Chain Reaction; Genetic Vectors; CRISPR-Cas Systems
PubMed: 38951186
DOI: 10.1007/s00253-024-13239-7 -
Archives of Microbiology Jun 2024A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth...
A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29 was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29 showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166 (96.8%) and Thioclava marina 11.10-0-13 (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29 formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29 and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29, the major fatty acids identified were summed feature 8 (Cω7c) and C and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29 included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29 comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29 and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29 represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29 (= KCTC 82,841 = MCCC 1K08523).
Topics: Seawater; Phylogeny; RNA, Ribosomal, 16S; Fatty Acids; DNA, Bacterial; Base Composition; China; Bacterial Typing Techniques; Phospholipids; Alphaproteobacteria; Sequence Analysis, DNA; Ubiquinone; Nucleic Acid Hybridization
PubMed: 38951168
DOI: 10.1007/s00203-024-04057-6 -
Methods in Molecular Biology (Clifton,... 2024Mathematical models have been used to study the spread of infectious diseases from person to person. More recently studies are developing within-host modeling which...
Mathematical models have been used to study the spread of infectious diseases from person to person. More recently studies are developing within-host modeling which provides an understanding of how pathogens-bacteria, fungi, parasites, or viruses-develop, spread, and evolve inside a single individual and their interaction with the host's immune system.Such models have the potential to provide a more detailed and complete description of the pathogenesis of diseases within-host and identify other influencing factors that may not be detected otherwise. Mathematical models can be used to aid understanding of the global antibiotic resistance (ABR) crisis and identify new ways of combating this threat.ABR occurs when bacteria respond to random or selective pressures and adapt to new environments through the acquisition of new genetic traits. This is usually through the acquisition of a piece of DNA from other bacteria, a process called horizontal gene transfer (HGT), the modification of a piece of DNA within a bacterium, or through. Bacteria have evolved mechanisms that enable them to respond to environmental threats by mutation, and horizontal gene transfer (HGT): conjugation; transduction; and transformation. A frequent mechanism of HGT responsible for spreading antibiotic resistance on the global scale is conjugation, as it allows the direct transfer of mobile genetic elements (MGEs). Although there are several MGEs, the most important MGEs which promote the development and rapid spread of antimicrobial resistance genes in bacterial populations are plasmids and transposons. Each of the resistance-spread-mechanisms mentioned above can be modeled allowing us to understand the process better and to define strategies to reduce resistance.
Topics: Bacteria; Humans; Gene Transfer, Horizontal; Drug Resistance, Microbial; Models, Theoretical; Drug Resistance, Bacterial; Anti-Bacterial Agents; Host-Pathogen Interactions
PubMed: 38949703
DOI: 10.1007/978-1-0716-3981-8_9 -
Mikrochimica Acta Jun 2024A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for...
A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/μL in a model plasmid containing the malB gene and 3 CFU/μL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.
Topics: Nucleic Acid Amplification Techniques; Escherichia coli; Limit of Detection; Milk; Animals; Molecular Diagnostic Techniques; Microfluidic Analytical Techniques; DNA, Bacterial
PubMed: 38949666
DOI: 10.1007/s00604-024-06509-8 -
Molecular Plant-microbe Interactions :... Jul 2024The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. pv. is one of the major pathogens causing bacterial...
The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. pv. is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of pv. , our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of pv. strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in pv. is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of pv. is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.
PubMed: 38949619
DOI: 10.1094/MPMI-04-24-0035-R