Abstract

The Staphylococcus aureus Recombinant plasmids pS194 and pSC194, which confer resistance to streptomycin and streptomycin-chloramphenicol, respectively, have been used as vectors for the construction of recombinant DNA, since each of them carries a unique receptor site for the EcoRI endonuclease.

The hybrid DNA does not express resistance to streptomycin, a marker that is present in both vectors, presumably because the marker gene is cut by EcoRI. A chloramphenicol marker present on pSC194 was used for positive hybrid selection. Hybrid plasmids generated by ligating pSC194 with one or more of the four EcoRI fragments of the large (18.1·106 daltons) staphylococcal plasmid p1258 were constructed and allowed us to develop a physical map to p1258.

Product name

Recombinant protein Staphylococcus aureus Staphylococcus Enterotoxin G (His tag)

Purity: > 90% SDS-PAGE.

Expression system: Escherichia coli

Accession: P0A0L7

Protein length: full length protein

Animal-free: No

Nature: recombinant

Species: staphylococcus aureus

Predicted Molecular Weight: 43 kDa including tags

Amino acids: 26 to 258

Tags: Your N-Terminus Tag

Additional sequence information

Strain N315. This product is the full-length mature protein from aa 26 to 258 with a 6xHis-SUMO tag at the N-terminus. The signal peptide is not included.

Specifications

Our Abpromise warranty covers the use of ab225609 in the following tested applications. Application notes include recommended starting dilutions; the end-user must determine the optimal dilutions/concentrations.

Applications: SDS PAGE

Form: Liquid

Concentration: 100 µg to 0.5 mg/mL

Stability and Storage

• Shipped at 4°C. At the time of aliquot delivery. Store at -20°C or -80°C. Avoid freeze/thaw cycle.
• Components: 50% glycerol (glycerin, glycerin), Tris buffer

Abstract

We have previously shown that substitution of the spike (S) gene of the apathogenic IBV strain Beau-R by that of the pathogenic strain of the same serotype, M41, resulted in an apathogenic virus, BeauR-M41(S), which conferred protection against challenge with M41. We have constructed a recombinant IBV, BeauR-4/91(S), with the genetic base of Beau-R but expressing the spike protein of the pathogenic strain IBV 4/91(UK), which belongs to a different serogroup such as Beaudette or M41.

Similar to our previous findings with BeauR-M41(S), observations of clinical signs showed that the S gene of Avian infectious bronchitis virus Recombinant 4/91 did not confer pathogenicity to rIBV BeauR-4/91(S). Furthermore, protection studies showed that there was homologous protection; BeauR-4/91(S) conferred protection against wild-type 4/91 virus challenge as evidenced by the absence of clinical signs, IBV RNA assessed by qRT-PCR, and the fact that no virus was isolated. from tracheas removed from birds primarily infected with BeauR-4/91(S) and challenged with IBV 4/91(UK).

A degree of heterologous protection against the M41 challenge was observed, albeit at a lower level. Our results confirm and extend our previous findings and conclusions that protein S ectodomain swapping is a precise and efficient way to generate genetically defined candidate IBV vaccines.

Statement of Ethics

All animal testing protocols were carried out in strict accordance with UK Home Office guidelines and the license granted for experiments involving regulated procedures on animals protected by the UK Animals (Scientific Procedures) Act. of 1986. The experiments were carried out at the IAH Ministry of the Interior under license (PCD30/4301) and were approved by the IAH ethical review committee under the terms of reference HO-ERP-01-1, using chickens obtained from the IAH Poultry Production Unit.

Cells and viruses

The pathogenic strain of IBV 4/91 (UK) used in this study was a gift from Intervet UK Ltd and was cultured in 10-day-old specific-pathogen-free (SPF) Rhode Island Red (RIR) embryonated hen eggs obtained from the Institutes. poultry production unit; Primary chicken kidney (CK) cells are refractory to the growth of IBV 4/91 (UK).

M41-CK was derived from the pathogenic IBV strain M41 after adaptation in CK cells. Vaccinia viruses (VV) were routinely grown and titrated in Vero cells as described previously, while large stocks for DNA isolation were prepared from infected BHK-21 cells. Tracheal organ cultures (TOC) were prepared from 19-day SPF RIR chick embryos. Virus infectivity titers were performed in TOC and titers were expressed as 50% (median) cytostatic dose (CD50).

Recovery of an infectious EBV expressing a chimeric S protein

CVV-BeauR-4/91(S) DNA was purified and initially used to rescue rIBV in CK cells. Cell lysate (0.1 ml) from the infected and transfected CK (P0) cells were used to infect 10 day SPF embryos. Infected embryos were incubated at 37°C for 48 h, after which they were placed at 4°C overnight. Allantoic fluid (EP1) was collected and passaged a further five times on 10 days old SPF embryos and the resulting rIBV, BeauR-4/91(S), was used in subsequent experiments.

RNA was extracted from the allantoic fluid of infected eggs using the RNeasy® method (Qiagen) for amplification of part of the S gene by RT-PCR (Ready-To-GoTM RT-PCR beads) to confirm the identity of EBV by sequence analysis. A stock of BeauR-4/91(S) was produced in 10 days old SPF embryonated eggs, final titer 2×105.6 CD50 per ml, which was used for subsequent in vivo experiments.

In vivo analysis of rIBVs

Virus stocks for in vivo experiments were prepared from 10 days old SPF embryonated RIR eggs and titrated by TOC; stock virus titers were 4/91(UK) 5.4 log10 CD50, BeauR-4/91(S) 5.6 log10 CD50, and M41-CK 6.0 log10 CD50 in a volume of 1 ml. Five groups (n = 13) of 8-day-old SPF RIR chickens were used for in vivo analysis of EBV BeauR-4/91(S). Chickens were housed in negative pressure, temperature-controlled, HEPA-filtered isolation rooms, with each group housed in a separate room.

Three groups of birds were inoculated conjunctival (eye drops) and intranasally with 3.6 log10 CD50 of BeauR-4/91(S) in 0.1 ml of serum-free BES (N, N-Bis(2-hydroxyethyl) -2- medium containing aminoethanesulfonic acid). The other two groups were inoculated with BES medium without serum as controls. Three weeks after infection, the three groups that had been infected with BeauR-4/91(S) were challenged using 3.6 log10 CD50 in a total of 0.1 ml with IBV 4/91(UK), IBV M41 -CK or mock-challenged and the two mock-infection groups were sham-challenged or 4/91 (UK); in all cases, challenge viruses were administered conjunctively and intranasally.

Pathogenicity assessment

The clinical signs used to determine pathogenicity were clicking (sneeze-like sound), tracheal rales (sound emanating from the bronchi, also detected by vibrations when holding a chick), wheezing (dyspnea), runny nose, watery eyes and ciliary tracheal activity. Chicks were observed daily for clinical signs; the snicks were counted independently by two people during a period of 2 min.

Birds were checked individually for the presence of tracheal rales, nasal discharge, watery eyes, and wheezing. Tracheas were removed from three randomly selected chickens from each group at 4, 5, and 6 days post-challenge to assess ciliary activity. Ten 1 mm sections were cut from three different regions of each trachea and the level of cryostasis was determined from each tracheal section using light microscopy.

Virus isolation

Tracheal sections stored in PBS were frozen, thawed, and homogenized using the Tissuelyser II (Qiagen). The resulting tracheal suspensions were centrifuged and the supernatants were used to infect TOC. Separate tracheal suspensions were prepared from three birds (except for mock-infected group: 4/91-challenged, n = 2) per sampling day (days 4, 5 and 6 post-challenge) for BeauR-4/91(S ):4/91 and BeauR-4/91(S): M41 groups.

Six TOCs were infected with 100 µl of the corresponding tracheal suspension. After infection at 37°C for 1 h, 0.5 ml of medium was added and the TOCs were incubated at 37°C for 7 days, during which they were regularly observed for ciliary activity. To compare ciliary activity results, ANOVA analysis followed by Dunnett’s posthoc multiple comparison test was performed using GraphPad Prism version 5.03.

Description

Filoviruses, Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines in development is a system based on recombinant vesicular stomatitis Indiana virus (rVSIV) that expresses an individual filovirus glycoprotein (GP) instead of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including rVSV filovirus GP vectors, is their safety. To address this concern, we conducted a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamicly. Seven animals received an rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received an rVSV vector expressing Lake Victoria marburgvirus (MARV) GP; three animals received rVSV wild-type (wt) vector and four animals received vehicle control.

Two of the three animals receiving rVSV-wt showed severe neurological symptoms, whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed significant lesions in the neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals in the filovirus vehicle or vaccine control groups. These data strongly suggest that the rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the parent rVSV-wt vector and support their further development as a vaccine platform for human use.

Purity: greater than 85% as determined by SDS-PAGE.

Destination Names: P

Uniprot No.: P04879

Research Area: Others

Alternative Names: Protein P; M1 protein

Species: Vesicular stomatitis Indiana virus (Glasgow strain) (VSIV)

Source: E.coli

Expression Region: 1-265aa

Mole Weight: 34.9 kDa

Protein Length: Total length

Tag information: N-terminal 10xHis-tagged and C-terminal Myc-tagged

Form: Liquid or Lyophilized Powder

Note: We will preferably ship the format we have in stock, however, if you have any special requirements for the format, please remark your requirement when placing the order, we will prepare according to your demand.

Buffer

If the dosage form is liquid, the default storage buffer is Tris/PBS based buffer, 5%-50% glycerol. If the administration form is a lyophilized powder, the buffer before lyophilization is Tris/PBS-based buffer, 6% trehalose, pH 8.0.

Reconstitution

We recommend that this vial be briefly centrifuged before opening to bring the contents to the bottom. Reconstitute protein in sterile deionized water at a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and an aliquot for long-term storage at -20°C/-80°C. Our final default glycerol concentration is 50%. Customers could use it for reference.

Storage Conditions

Store at -20°C/-80°C upon receipt, need to be aliquoted for multiple uses. Avoid repeated cycles of freezing and thawing.

Shelf life

Shelf life is related to many factors, storage condition, buffer ingredients, storage temperature and the stability of the protein itself. Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.

Delivery time: 3-7 business days

Notes: Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.

Materials And Methods

• Animals

A total of 21 healthy male cynomolgus macaques (Macaca fascicularis) (4-7 kg) were purchased from Charles River Laboratories (Wilmington, MA). All animals were between 4 and 6 years of age with the exception of two animals (67-01, 68-01) that were 18 years old. The study was conducted at the New England Primate Research Center (PRC), Harvard Medical School, and the animals were cared for in accordance with the standards of the Association for the Evaluation and Accreditation of Laboratory Animal Care. and the Harvard Medical School Animal Care and Use Committee.

All animal work adhered to the regulations outlined in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press) and by the Harvard Medical School Animal Care and Use Committee. These experiments and procedures were approved by the Harvard Medical Area Standing Committee on Animals. Any clinical signs of illness or distress were immediately reported to the responsible veterinarian, who recommended treatment of minor ailments or euthanasia when clinical observations and neurological scores of the animals reached levels based on the protocol approved by the Care Committee. and Use of Animals from Harvard Medical School.

• Haematology and serum biochemistry

Total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, mean cell volume, mean corpuscular volume, and mean corpuscular haemoglobin concentration were determined from Blood samples collected in tubes containing EDTA, using a laser-based haematology analyzer (Hemavet, Drew Scientific, Waterbury, CT).

Albumin, amylase, globulin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, lactate dehydrogenase, glucose, cholesterol, total protein, total bilirubin, direct bilirubin, urea nitrogen, creatinine, creatinine kinase, triglycerides, bicarbonate, calcium, phosphorus, chloride, potassium, and sodium were measured by IDEXX VetConnect (Westbrook, ME) using an Olympus AU5421 chemistry analyzer (Olympus Americas, Center Valley, PA).

• Necropsy and tissue collection

Animals were euthanized if neurological signs developed or at the scheduled study endpoint, 21 days after inoculation, with an overdose of intravenous sodium pentobarbital and necropsied immediately thereafter. Tissues from five brain regions (frontal cortex (FC), occipital cortex (OC), cerebellum (CB), thalamus (TH), and basal ganglia (BG)) and three spinal cord (SC) regions (cervical, thoracic, and lumbar ) were collected in 10% neutral buffered formalin. After one week of fixation, the brain was sectioned into right and left hemispheres and specific brain regions were trimmed and embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. (H&E).

• Isolation of VSV

To assess whether there was virus replication in nasal, oral, and rectal swabs (days 0, 2, 4, 7, 14, and 21), blood (days 0, 2, 4, 7, 14, and 21), non-neural tissues (spleen, liver, kidney, heart, axillary lymph node and adrenal gland) and neural tissues (FC, BG, OC, TH, CB, BS and SC) virus titers were determined by standard plaque assay on Vero monolayer cells. Swabs were collected and plated in D-10 medium, while tissues were plated in D-10 medium and then homogenized prior to plating assay.