Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA

Background: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA.
Methods: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels.
Results: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs.
Conclusions: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.
Keywords: Hexahistidine tag; Isothermal DNA amplification; Recombinase polymerase amplification (RPA); uvsY.

A Strategy to Fight against Triple-Negative Breast Cancer: pH-Responsive Hexahistidine-Metal Assemblies with High-Payload Drugs

Triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer, is difficult to be targeted therapeutically due to negative expression of the bioreceptor, which leads to the poorest overall four-year survival rate among all cancer subtypes.
We proposed that the nanomedicine featuring high payload and pH-responsive release of the loaded drugs could assist the TNBC treatment. In the present study, the His6-metal assemblies (HmA) were employed to encapsulate the doxorubicin (Dox), and the effect of HmA loaded with Dox (HmA@Dox) on treating TNBC was evaluated in vitro and in vivo.
We found that the participation of Dox in the formation of HmA leads to high loading efficiency (99.4% for concentration ≤ 1 mg/mL) and the loading capacity (50.7% for concentration ≥ 10 mg/mL) of Dox encapsulated into HmA. HmA@Dox exhibited a narrow size distribution on the nanoscale, a pH-responsive release of loaded Dox, a quick endocytosis process, and fast lysosome escape. Most importantly, the HmA@Dox showed high efficacy in killing various breast cancer cells (MCF-7, MDA-MB-231, and MDA-MB-453) in vitro and depressing the development of TNBC in vivo.
Our results demonstrated that such a strategy for designing a nanomedicine with high payload and responsive release of drugs to the environment around the tumor was of great importance to treat TNBC.

Efficient Delivery of Antibodies Intracellularly by Co-Assembly with Hexahistidine-Metal Assemblies (HmA)

Purpose: There has been a substantial global market for antibodies, which are based on extracellular targets. Binding intracellular targets by antibodies will bring new chances in antibody therapeutics and a huge market increase. We aim to evaluate the efficiency of a novel delivery system of His6-metal assembly (HmA) in delivering intracellular antibodies and biofunctions of delivered antibodies.
Methods: In this study, the physicochemical properties of HmA@Antibodies generated through co-assembling with antibodies and HmA were well characterized by dynamic light scatter. The cytotoxicity of HmA@Antibodies was investigated by Cell Counting Kit-8 (CCK-8). The endocytic kinetics and lysosome escape process of HmA@Antibodies were studied by flow cytometry and fluorescent staining imaging, respectively. Compared to the commercialized positive control, the intracellular delivery efficiency by HmA@Antibodies and biofunctions of delivered antibodies were evaluated by fluorescent imaging and CCK-8.
Results: Various antibodies (IgG, anti-β-tubulin and anti-NPC) could co-assemble with HmA under a gentle condition, producing nano-sized (~150 nm) and positively charged (~+30 eV) HmA@Antibodies particles with narrow size distribution (PDI ~ 0.15). HmA displayed very low cytotoxicity to divers cells (DCs, HeLa, HCECs, and HRPE) even after 96 h for the feeding concentration ≤100 μg mL-1, and fast escape from endosomes. In the case of delivery IgG, the delivery efficiency into alive cells of HmA was better than a commercial protein delivery reagent (PULSin).
For cases of the anti-β-tubulin and anti-NPC, HmA showed comparable delivery efficiency to their positive controls, but HmA with ability to deliver these antibodies into alive cells was still superior to positive controls delivering antibodies into dead cells through punching holes.
Conclusion: Our results indicate that this strategy is a feasible way to deliver various antibodies intracellularly while preserving their functions, which has great potential in various applications and treating many refractory diseases by intracellular antibody delivery.
Keywords: antibody; coordination polymer; intracellular delivery; nanocarrier; peptide assembly.

Efficient delivery of cytosolic proteins by protein-hexahistidine-metal co-assemblies

Proteins play key roles in most biological processes, and protein dysfunction can cause various diseases. Over the past few decades, tremendous development has occurred in the protein therapeutic market due to the high specificity, low side effects, and low risk of proteins.
Currently, all protein drugs on the market are based on extracellular targeting; more than 70% of intracellular targets remain un-druggable. Efficient delivery of cytosolic proteins is of significance for protein drugs, advanced biotechnology and molecular cell biology. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for intracellular protein delivery.
Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged protein encapsulation particles(Protein@HmA) through a co-assembly process with a high loading capacity and loading efficiency.
Protein@HmA was able to deliver proteins with diverse physicochemical properties through multiple endocytosis pathways, and the protein could quickly escape from endosomes.
In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells. Our results demonstrate that this strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics,
 STATEMENT OF SIGNIFICANCE: : Intracellular targets with protein drugs may provide a new way for the treatment of many refractory disease. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for efficient intracellular protein delivery.

Anti-Hexahistidine (His6) tag Antibody

STJ60100 St John's Laboratory 100 µg 424 EUR

Recombinant Human MBL2 Protein-Hexahistidine tag

CTP-244 Creative Biolabs 100ug Ask for price
Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged particles (Protein@HmA) with a high loading efficiency.
Protein@HmA was able to deliver different proteins through multiple endocytosis pathways, and the protein could quickly escape from endosomes. In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells.
This strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics.

Using a Syrian (Golden) Hamster Biological Model for the Evaluation of Recombinant Anthrax Vaccines

In this paper, we demonstrate that a Syrian hamster biological model can be applied to the study of recombinant anthrax vaccines. We show that double vaccination with recombinant proteins, such as protective antigen (PA) and fusion protein LF1PA4, consisting of lethal factor I domain (LF) and PA domain IV, leads to the production of high titers of specific antibodies and to protection from infection with the toxicogenic encapsulated attenuated strain B. anthracis 71/12.
In terms of antibody production and protection, Syrian hamsters were much more comparable to guinea pigs than mice. We believe that Syrian hamsters are still underestimated as a biological model for anthrax research, and, in some cases, they can be used as a replacement or at least as a complement to the traditionally used mouse model.

Vaccination with a Brucella ghost developed through a double inactivation strategy provides protection in Guinea pigs and cattle

Vaccination can prevent and control animal brucellosis. Currently, live attenuated vaccines are extensively used to prevent Brucella infection. However, traditional vaccines such as live attenuated vaccines are associated with biological safety risks for both humans and animals. The bacterial ghost (BG) is a new form of vaccine with great prospects. However, bacterial cells cannot be completely inactivated by biological lysis https://www.joplink.net/guinea-pig-antibodies/, conferring a safety risk associated with the vaccine.
In this study, we developed a Brucella abortus A19 bacterial ghost (A19BG) through a double inactivation strategy with sequential biological lysis and hydrogen peroxide treatment. This strategy resulted in 100% inactivation of Brucella, such that viable bacterial cells were not detected even at an ultrahigh concentration of 1010 colony-forming units/mL. Furthermore, A19BG had a typical BG morphology and good genetic stability. Moreover, it did not induce adverse reactions in guinea pigs.
The levels of antibodies, interferon-γ, interleukin-4, and CD4+ T cells in guinea pigs inoculated with the A19BG vaccine were similar to those inoculated with the existing A19 vaccine. Immunization with A19BG conferred a similar level of protection with that of A19 against Brucella melitensis M28 in both guinea pigs and cattle. In conclusion, the combination of biological lysis and H2O2-mediated inactivation is a safe and effective strategy that can serve as a reference for the preparation of BG vaccines.

Infection and protection responses of deletion mutants of non-structural proteins of foot-and-mouth disease virus serotype Asia1 in guinea pigs

The development of a negative marker vaccine against the foot-and-mouth disease virus (FMDV) will enhance the capabilities to differentiate vaccinated from infected animals and move forward in the progressive control pathway for the control of FMD. Here, we report the development of mutant FMDV of Asia1 with partial deletion of non-structural proteins 3A and 3B and characterization of their infectivity and protection response in the guinea pig model. The deleted FMDV Asia1/IND/63/1972 mutants, pAsiaΔ3A and pAsiaΔ3A3B1 were constructed from the full-length infectious clone pAsiaWT, the viable virus was rescued, and the genetic stability of the mutants was confirmed by 20 monolayer passages in BHK21 cells.
The mutant Asia1 viruses showed comparable growth pattern and infectivity with that of AsiaWT in the cell culture. However, the AsiaΔ3A3B1 virus showed smaller plaque and lower virus titer with reduced infectivity in the suckling mice. In guinea pigs, the AsiaΔ3A3B1 virus failed to induce the disease, whereas the AsiaΔ3A virus induced typical secondary lesions of FMD. Vaccination with inactivated Asia1 mutant viruses induced neutralizing antibody response that was significantly lower than that of the parent virus on day 28 post-vaccination (dpv) in guinea pigs (P < 0.05).
Furthermore, challenging the vaccinated guinea pigs with the homologous vaccine strain of FMDV Asia1 conferred complete protection. It is concluded that the mutant AsiaΔ3A3B1 virus has the potential to replace the wild-type virus for use as a negative marker vaccine after assessing the vaccine worth attributes in suspension cell and protective efficacy study in cattle.Key points• Deletion mutant viruses of FMDV Asia1, developed by PCR-mediated mutagenesis of NSP 3A and 3B1, were genetically stable.• The growth kinetics and antigenic relatedness of the mutant viruses were comparable with that of the wild-type virus.• Vaccination of guinea pigs with the deletion mutant viruses conferred complete protection upon challenge with the homologous virus.

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms.
salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays.
This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

Characterization of Canine Influenza Virus A (H3N2) Circulating in Dogs in China from 2016 to 2018

Avian H3N2 influenza virus follows cross-host transmission and has spread among dogs in Asia since 2005. After 2015-2016, a new H3N2 subtype canine influenza epidemic occurred in dogs in North America and Asia. The disease prevalence was assessed by virological and serological surveillance in dogs in China.
Herein, five H3N2 canine influenza virus (CIV) strains were isolated from 1185 Chinese canine respiratory disease samples in 2017-2018; these strains were on the evolutionary branch of the North American CIVs after 2016 and genetically far from the classical canine H3N2 strain discovered in China before 2016. Serological surveillance showed an HI antibody positive rate of 6.68%. H3N2 was prevalent in the coastal areas and northeastern regions of China. In 2018, it became the primary epidemic strain in the country.
The QK01 strain of H3N2 showed high efficiency in transmission among dogs through respiratory droplets. Nevertheless, the virus only replicated in the upper respiratory tract and exhibited low pathogenicity in mice. Furthermore, highly efficient transmission by direct contact other than respiratory droplet transmission was found in a guinea pig model.
The low-level replication in avian species other than ducks could not facilitate contact and airborne transmission in chickens. The current results indicated that a novel H3N2 virus has become a predominant epidemic strain in dogs in China since 2016 and acquired highly efficient transmissibility but could not be replicated in avian species. Thus, further monitoring is required for designing optimal immunoprophylactic tools for dogs and estimating the zoonotic risk of CIV in China.

Hemoglobin Antibody

  • 317.00 EUR
  • 335.00 EUR
  • 100ug
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Hemoglobin Antibody

  • 317.00 EUR
  • 335.00 EUR
  • 100ug
  • 50ug

Hemoglobin Antibody

  • 317.00 EUR
  • 335.00 EUR
  • 100ug
  • 50ug

Hemoglobin Antibody

  • 317.00 EUR
  • 335.00 EUR
  • 100ug
  • 50ug

Hemoglobin Antibody

  • 317.00 EUR
  • 335.00 EUR
  • 100ug
  • 50ug

Hemoglobin Antibody

1 mg 537 EUR

Hemoglobin Antibody

1 mg 537 EUR

Hemoglobin antibody

100 ul 393 EUR

Hemoglobin antibody

1 mg 343 EUR

Hemoglobin antibody

1 mg 30 EUR