ORFs coding for Nefmut fused with S1, S2, M, or N SARS-CoV-2 proteins were cloned into pVAX1 plasmid (Thermo Fisher, Waltham, MA, USA), i.e., a vector approved by FDA for use in humans. To obtain the pVAX1 vector expressing Nefmut, its ORF was cloned into Nhe I and EcoR I sites. To recover vectors expressing Nefmut-based fusion products, an intermediate vector referred to as pVAX1/Nefmutfusion was produced. Here, the whole Nefmut ORF deprived of its stop codon was followed by a sequence coding a GPGP linker including a unique Apa I restriction site. In this way, sequences comprising the Apa I site at their 5′ end, and the Pme I one at their 3′ end were fused in frame with Nefmut ORF. Stop codons of SARS-CoV-2-related sequences were preceded by sequences coding for a DYKDDDK epitope tag (flag-tag). SARS-CoV-2 sequences were optimized for expression in human cells through GeneSmart software from Genescript. All these vectors were synthesized by Explora Biotech. The pTargeT vector expressing the Nefmut/HPV16-E7 fusion protein was already described [38 (link)].
Pvax1 plasmid
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PVAX1 plasmid is a circular DNA molecule used in molecular biology and biotechnology applications. It serves as a vector for the propagation and expression of genetic sequences in host cells. The core function of the PVAX1 plasmid is to provide the necessary elements, such as a promoter and selectable markers, to facilitate the cloning and manipulation of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Endofree plasmid giga kit, by Qiagen (2 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
T7 ribomax express large scale rna production system, by Promega (1 mentions)
Superscript 3 platinum one step qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
9 protocols using pvax1 plasmid
1
SARS-CoV-2 Fusion Protein Expression
2
Nef-SARS-CoV-2 fusion protein constructs
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Open-reading frames coding for Nefmut fused with either S1, S2, or N SARS-CoV-2 proteins were cloned into pVAX1 plasmid (Thermo Fisher, Waltham, MA, USA), as previously described [18 (link)]. In these constructs, S1 spans from aa 19, i.e., downstream of the signal peptide, to aa 680, just upstream of the furin-like cleavage site; S2 included the extracellular portion of the subunit with the exclusion of the two fusion domains; and finally, the entire N protein (422 aa), except M1 amino acid, was fused to Nefmut. A GPGP linker was inserted between Nefmut and the downstream sequences. Stop codons of SARS-CoV-2-related sequences were preceded by sequences coding for a DYKDDDK epitope tag (flag-tag). SARS-CoV-2 sequences were optimized for expression in human cells through GenSmart™ Codon Optimization software from Genescript. All vectors were synthesized by Explora Biotech (Venice, Italy).
3
Cloning and Expression of Mutant Nef Variants
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Nefmut, Nefmut/N and NefmutPL open reading frames (ORFs) were cloned into pVAX1 plasmid (Thermo Fisher, Waltham, MA, USA), i.e., a vector approved by FDA for use in humans, as previously described [14 (link),15 (link),16 (link)]. ORFs coding for NefmutPL fused with SARS-CoV-2 N protein was cloned into pVAX1 plasmid as well. To this aim, an intermediate vector referred to as pVAX1/Nefmutfusion was used [18 (link)]. Here, the NefmutPL ORF deprived of its stop codon was followed by a sequence coding a GPGP linker, including a unique Apa I restriction site. In this way, sequences comprising the Apa I site at their 5′ end, and the Pme I one at their 3′ end were fused in frame with NefmutPL ORF. Stop codons was preceded by sequences coding for a DYKDDDK epitope tag (flag-tag). The vector was synthesized by Explora Biotech.
4
Nef Mutant SARS-CoV-2 Fusion Protein Expression
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ORFs coding for Nef mut fused with S1, S2, M, and N SARS-CoV-2 proteins were cloned into pVAX1 plasmid (Thermo Fisher), i.e., a vector approved by FDA for use in humans. To obtain the pVAX1 vector expressing Nef mut , its ORF was cloned into Nhe I and EcoR I sites (Fig. 1A). To recover vectors expressing Nef mut -based fusion products, an intermediate vector referred to as pVAX1/Nef mut fusion was produced (Fig. 1A). Here, the whole Nef mut ORF deprived of its stop codon was followed by a sequence coding a GPGP linker including a unique Apa I restriction site. In this way, sequences comprising the Apa I site at their 5' end and the Pme I one at their 3' end were fused in frame with Nef mut ORF (Fig. 1). Stop codons of SARS-CoV-2-related sequences were preceded by sequences coding for a DYKDDDK epitope tag (flag-tag). SARS-CoV-2 sequences were optimized for expression in human cells through the GeneSmart software from Genescript. All these vectors were synthesized by Explora Biotech. The pTargeT vector expressing the Nef mut /HPV16-E7 fusion protein was already described (74) (link).
5
Immunization with mROS1 and hROS1 Plasmids
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The plasmids coding the extracellular and transmembrane domain of the mouse (m) and the human (h) ROS1 cDNA sequences were obtained from GenScript (Piscataway, NJ, USA). Regarding the mROS1 plasmid, the cDNA sequence inserted was optimized in order to enhance its immunogenicity in mice. The ROS1 protein sequence coded by the hROS1 plasmid shows a homology of 80% with the mROS1 protein. Both sequences were cloned in pVAX1 plasmid (Invitrogen), sequenced (BMR Genomics, Padova, Italy), and amplified with EndoFree Plasmid Giga Kits (Qiagen Inc., Hilden, Germany). Before each immunization, mice were anaesthetized, as described above, and then their quadriceps muscle was injected with 50 μg of either pVAX, or mROS1 or hROS1 plasmids diluted in 20 μL of saline solution. Immediately after the injection, two 25-ms transcutaneous low-voltage electric pulses (amplitude 150 V; interval 300 ms) were administered at the injection site via a multiple-needle electrode connected to a CliniporatorTM (IGEA) [74 (link),75 (link),76 (link)]. For transplantable tumor growth experiments, wt mice were immunized twice at 14-day intervals, while, as far as the transgenic mouse model is concerned, 6-week-old K-RasG12D mice were randomly assigned to pVAX, mROS1, and hROS1 experimental groups and immunized three times at 14-day intervals.
6
SARS-CoV-2 RdRp Gene Quantification
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The coding sequence of SARS-CoV-2 RdRp gene was chemically synthesized and cloned into pVAX1 plasmid (Invitrogen, Carlsbad, CA, USA) between KpnI and NotI restriction sites. The plasmid was propagated in DH5α cells and purified using MiniPrep Qiagen Kits (Qiagen, Manchester, UK). The linearized plasmid with pVAX1-RdRP was used for in vitro transcription using the T7 RiboMAX™ Express Large-Scale RNA Production System (Promega, Madison, WI, USA). The copy number of in vitro transcribed RNA was calculated from RNA concentration measured with NanoDrop™ 2000c Spectrophotometers (Thermo-Fisher Scientific, Waltham, MA, USA) in triplicate. RNA products were then purified using the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA). A standard curve was generated using dilutions of the standard in vitro transcribed RNAs using SuperScript III Platinum One-Step qRT-PCR Kit as per the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA) using the CFX96 Touch Real-Time PCR Detection System (BioRad Laboratories, Watford, UK).
7
Truncated ORF2 Subcloning and Expression
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Escherichia coli strain DH5α (Novagen Inc., Madison, Wis., USA) was used for transformation and the pVAX1 plasmid (Invitrogen®, Carls-bad, CA, USA) was used for subcloning of the truncated ORF2 and tPAsp-PADRE-truncated ORF2 (112 - 660 aa) gene cassette. Eukaryotic expression vector, pVAX1, was constructed to be consistent with the Food and Drug Administration (FDA) regulations. HEK293 cells, routinely maintained in our laboratory, and CHO cell line, purchased from the national cell bank of Iran (Pasteur Institute), were used for protein expression. All kits were purchased from the Qiagen company (Germany); T4 DNA ligase and restriction endonucleases were purchased from New England Biolabs (New England Biolabs Inc., USA). All chemicals were purchased from Merck (Germany) and Sigma-Aldrich Corporation (Germany).
8
Construction of FILIP1LΔC103-p Plasmid
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pVAX1 plasmid (Invitrogen, San Diego, CA) encoding mutant-type FILIP1L (FILIP1LΔC103) named FILIP1LΔC103-p was constructed in our laboratory as described in a previous study [2 (link)]. The pVAX1 plasmid without FILIP1LΔC103 (named E-p) was used as an empty-vector control. Large-scale plasmid DNA was purified using an EndoFree Plasmid Giga kit (Qiagen). The DNA was dissolved in sterile endotoxin-free water and adjusted eventually to 1.0 mg/ml, and then stored at -20°C for the future use.
9
Enhancing Eukaryotic Expression of Vaccine Sequence
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In order to make the vaccine sequence better expressed in eukaryotes, we sequentially added the Kozak sequence containing the start codon (GCCACCATG ) and a piece of IgE leader sequence (DWTWILFLVAAATRVHS ) at its 5’ end. The JCat codon optimization tool42 (link) (http://www.jcat.de/ ) was utilized for reverse translation and codon optimization of BD3-12P. The codon-optimized sequence synthesized by Shanghai Generay Biotech, was inserted into the pVAX1 plasmid (Invitrogen, 35–1049) between the EcoRI and XbaI sites.
Female C57BL/6 mice were procured and housed in a pathogen-free environment at the Institute of Medical Biology, Chinese Academy of Medical Sciences. The Institute of Medical Biology’s Institutional Animal Ethics Committee approved all animal-based experiments (Approval number: DWSP202306014). TC-1 tumor cells, originating from C57BL/6 mice primary lung epithelial cells and transformed with c-Ha-ras and HPV-16 E6- and E7-encoding genes,43 (link) served as a preclinical tumor model for assessing therapeutic HPV vaccines targeting HPV16 E6/E7 antigens.
Female C57BL/6 mice were procured and housed in a pathogen-free environment at the Institute of Medical Biology, Chinese Academy of Medical Sciences. The Institute of Medical Biology’s Institutional Animal Ethics Committee approved all animal-based experiments (Approval number: DWSP202306014). TC-1 tumor cells, originating from C57BL/6 mice primary lung epithelial cells and transformed with c-Ha-ras and HPV-16 E6- and E7-encoding genes,43 (link) served as a preclinical tumor model for assessing therapeutic HPV vaccines targeting HPV16 E6/E7 antigens.
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