Limits...
Selection of anti-sulfadimidine specific ScFvs from a hybridoma cell by eukaryotic ribosome display.

Qi Y, Wu C, Zhang S, Wang Z, Huang S, Dai L, Wang S, Xia L, Wen K, Cao X, Wu Y, Shen J - PLoS ONE (2009)

Bottom Line: The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning.The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.

View Article: PubMed Central - PubMed

Affiliation: National Center for Veterinary Drug Safety Evaluation, College of Veterinary Medicine, China Agricultural University, Beijing, China.

ABSTRACT

Background: Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes.

Methodology/principal findings: In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA-ribosome-antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM(2) by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.

Conclusions/significance: The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.

Show MeSH

Related in: MedlinePlus

Construction of the template of ribosome display.In this generalized construct for ribosome display, the gene of interest is flanked by a 5′sequence with the T7 promoter (T7) and eukaryotic translation initiation (Kozak) sequence, and a tolA domain is attached at the 3′end as a spacer. The stop codon is removed to ensure stalling of the ribosome at the end of translation. X indicates removal of the stop codon.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2712767&req=5

pone-0006427-g006: Construction of the template of ribosome display.In this generalized construct for ribosome display, the gene of interest is flanked by a 5′sequence with the T7 promoter (T7) and eukaryotic translation initiation (Kozak) sequence, and a tolA domain is attached at the 3′end as a spacer. The stop codon is removed to ensure stalling of the ribosome at the end of translation. X indicates removal of the stop codon.

Mentions: Total RNA was extracted from 107 Hybridoma cells produced previously by He et al [34] that secreted monoclonal antibody against SM2 by using SV Total RNA Isolation System (Promega, USA). About 0.5 µg of RNA was reverse transcribed by Oligo dT-Adaptor primers of the first-strand cDNA synthesis kit (TaKaRa, JAPAN). cDNA encoding the mouse variable heavy (VH) and light (VL) chains were amplified by RT-PCR with degenerated immunoglobulin PCR primers (35 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min). A complementary (Gly4Ser)3-linker was added by a re-amplification. The full-length scFvs were assembled by using splicing overlap extension PCR. The purified products were cloned into the vector pRDV to add a 5′-T7-promotor and ribosome binding site and 3′-spacer region (Fig. 6). The templates of ribosome display were amplified directly from the ligation mixture [35]. The primers used in the PCR amplification were shown in Table 1.


Selection of anti-sulfadimidine specific ScFvs from a hybridoma cell by eukaryotic ribosome display.

Qi Y, Wu C, Zhang S, Wang Z, Huang S, Dai L, Wang S, Xia L, Wen K, Cao X, Wu Y, Shen J - PLoS ONE (2009)

Construction of the template of ribosome display.In this generalized construct for ribosome display, the gene of interest is flanked by a 5′sequence with the T7 promoter (T7) and eukaryotic translation initiation (Kozak) sequence, and a tolA domain is attached at the 3′end as a spacer. The stop codon is removed to ensure stalling of the ribosome at the end of translation. X indicates removal of the stop codon.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712767&req=5

pone-0006427-g006: Construction of the template of ribosome display.In this generalized construct for ribosome display, the gene of interest is flanked by a 5′sequence with the T7 promoter (T7) and eukaryotic translation initiation (Kozak) sequence, and a tolA domain is attached at the 3′end as a spacer. The stop codon is removed to ensure stalling of the ribosome at the end of translation. X indicates removal of the stop codon.
Mentions: Total RNA was extracted from 107 Hybridoma cells produced previously by He et al [34] that secreted monoclonal antibody against SM2 by using SV Total RNA Isolation System (Promega, USA). About 0.5 µg of RNA was reverse transcribed by Oligo dT-Adaptor primers of the first-strand cDNA synthesis kit (TaKaRa, JAPAN). cDNA encoding the mouse variable heavy (VH) and light (VL) chains were amplified by RT-PCR with degenerated immunoglobulin PCR primers (35 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min). A complementary (Gly4Ser)3-linker was added by a re-amplification. The full-length scFvs were assembled by using splicing overlap extension PCR. The purified products were cloned into the vector pRDV to add a 5′-T7-promotor and ribosome binding site and 3′-spacer region (Fig. 6). The templates of ribosome display were amplified directly from the ligation mixture [35]. The primers used in the PCR amplification were shown in Table 1.

Bottom Line: The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning.The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.

View Article: PubMed Central - PubMed

Affiliation: National Center for Veterinary Drug Safety Evaluation, College of Veterinary Medicine, China Agricultural University, Beijing, China.

ABSTRACT

Background: Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes.

Methodology/principal findings: In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA-ribosome-antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM(2) by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.

Conclusions/significance: The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.

Show MeSH
Related in: MedlinePlus