Manuel Gutiérrez-Rodríguez (1923-2009):A cytohistochemist from Cadiz

Herrera-Rodríguez, Francisco; Sarasquete, Carmen

doi:10.3989/scimar.2009.73n3617

Cited by 2 publications

(6 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…3). A previous hypothesis suggests that the glutamic acid-lysine repeat creates a specific environment on the VLP surface able to interact with the metal matrix [31]. Epitope insertion within or near this repeat may change the particular outside environment of the VLP, negatively affecting its affinity to the matrix.…”

Section: Expression and Purification Of Chimeric Prsv Particlesmentioning

confidence: 98%

“…We designed two sets of primers (Table 1) to generate two PRSV-CP genes with the PCV2b-224 epitope (FNLKDPPLNP) inserted at the N-or C-termini (PRSV-PCV2-N and PRSV-PCV2-C, respectively) by sequential PCRs. We used a previously described expression plasmid as the template, which harbors the native CP gene from a PRSV isolate [31]. Simultaneously, we designed a PRSV-CP chimeric gene (PRSV-PCV2 [19][20][21][22][23][24][25][26][27][28] ) with the insertion of the PCV2 epitope PCV2b-224 at a site of high antigenicity, according to an analysis carried out with CLC Main Workbench 6.0 (CLC Bio, Waltham, MA, USA).…”

Section: Construction Of Prsv-pcv2 Expression Vectorsmentioning

confidence: 99%

“…We transformed electrocompetent E. coli One Shot BL21Star (DE3) cells (Thermo Fisher Scientific, Carlsbad, CA, USA) with each plasmid. Chimeric CP expression was carried out as per Guerrero-Rodriguez et al [31] with some modifications. Briefly, a fresh colony was taken from an LB medium agar plate, placed in 3 mL of liquid LB/kanamycin medium (50 μg/mL), and incubated overnight at 37 • C and 250 rpm.…”

Section: Protein Expression and Particle Purificationmentioning

confidence: 99%

“…Total protein lysate was centrifuged (4 • C, 20 Min, and 2,370 × g), and supernatants were analyzed by SDS-PAGE and Coomassie blue staining. We carried out immobilized metal affinity chromatography as described elsewhere [31]. Alternatively, for chimeric VLPs purification, samples were treated with 4% (w/v) polyethylene glycol (PEG) 8000 for 1.5 H at 4 • C with constant shaking and left for 1 H at room temperature (22 ± 2 • C).…”

Section: Protein Expression and Particle Purificationmentioning

confidence: 99%

“…Expression of the chimeric PRSV CPs in E. coli was carried out according to a previous work [31]. We analyzed the ex-pression in IPTG-induced cultures by SDS-PAGE Coomassie blue staining.…”

Section: Expression and Purification Of Chimeric Prsv Particlesmentioning

confidence: 99%

See 4 more Smart Citations

Porcine circovirus type 2 protective epitope densely carried by chimeric papaya ringspot virus–like particles expressed in Escherichia coli as a cost‐effective vaccine manufacture alternative

Aguilera

Chávez-Calvillo

Elizondo-Quiroga

et al. 2016

Biotech and App Biochem

View full text Add to dashboard Cite

Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus-like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self-assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N- and C-termini and a predicted antigenic region located near the N-terminus. Wild-type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope-presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small-scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2.

show abstract

Section: Expression and Purification Of Chimeric Prsv Particlesmentioning

confidence: 98%

Section: Construction Of Prsv-pcv2 Expression Vectorsmentioning

confidence: 99%

Section: Protein Expression and Particle Purificationmentioning

confidence: 99%

Section: Protein Expression and Particle Purificationmentioning

confidence: 99%

“…Expression of the chimeric PRSV CPs in E. coli was carried out according to a previous work [31]. We analyzed the ex-pression in IPTG-induced cultures by SDS-PAGE Coomassie blue staining.…”

Section: Expression and Purification Of Chimeric Prsv Particlesmentioning

confidence: 99%

See 3 more Smart Citations