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Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

Li YL, Ma ZL, Zhao Y, Zhang J - Oncol Lett (2015)

Bottom Line: The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside.Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control.These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Shanghai University, Shanghai 200444, P.R. China ; Institute of Molecular Medicine, Nanjing University, Nanjing, Jiangsu 210093, P.R. China.

ABSTRACT

Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10(5) TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

No MeSH data available.


Related in: MedlinePlus

Construction of the pET-28a(+)-mE7 plasmid. The HPV16 mE7 gene was amplified by splicing overlap extension PCR using pET-28a(+)-E7 as a template. The mE7 gene was then directly inserted into a pMD18T vector to form pMD18T-mE7. The pET-28a(+) and pMD18T-mE7 vectors were each digested with restriction endonucleases NdeI and HindIII. Subsequent to purification with electrophoresis in a 2% agarose gel, mE7 and pET-28a(+) were ligated with T4 DNA ligase to yield the expression plasmid pET-28a(+)-mE7. PCR, polymerase chain reaction; HPV, human papillomavirus; ori, origin of replication; mE7, mutant non-transforming HPV16 E7.
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f1-ol-09-04-1851: Construction of the pET-28a(+)-mE7 plasmid. The HPV16 mE7 gene was amplified by splicing overlap extension PCR using pET-28a(+)-E7 as a template. The mE7 gene was then directly inserted into a pMD18T vector to form pMD18T-mE7. The pET-28a(+) and pMD18T-mE7 vectors were each digested with restriction endonucleases NdeI and HindIII. Subsequent to purification with electrophoresis in a 2% agarose gel, mE7 and pET-28a(+) were ligated with T4 DNA ligase to yield the expression plasmid pET-28a(+)-mE7. PCR, polymerase chain reaction; HPV, human papillomavirus; ori, origin of replication; mE7, mutant non-transforming HPV16 E7.

Mentions: The procedure for the construction of the pET-28a(+)-mE7 plasmid is shown in Fig. 1. The HPV16 mE7 gene was amplified by splicing PCR using pET-28a(+)-E7 as a template. The mE7 gene was then directly inserted into a pMD18T vector to form the pMD18T-mE7 plasmid. The pET-28a(+) and pMD18T-mE7 vectors were each digested with the NdeI and HindIII restriction endonucleases. Subsequent to purification by electrophoresis on a 2% agarose gel, mE7 and pET-28a(+) were ligated with T4 DNA ligase to yield the pET-28a(+)-mE7 expression plasmid. Compared with the E7 gene, mE7 was lacking nucleotides 280–294, which corresponds to residues 94–98 in the E7 protein, and nucleotide 70 in mE7 is changed from T to G, which results in residue 24 in the E7 protein being changed from Cys to Gly. The construction of the pET-28a(+)-mE7 plasmid allowed the expression of the fusion mE7 protein to have a hexa-histidine tag (his-tag) at the N-terminal end. The his-tag sequence served as a tag for affinity purification using a Ni-NTA agarose column.


Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

Li YL, Ma ZL, Zhao Y, Zhang J - Oncol Lett (2015)

Construction of the pET-28a(+)-mE7 plasmid. The HPV16 mE7 gene was amplified by splicing overlap extension PCR using pET-28a(+)-E7 as a template. The mE7 gene was then directly inserted into a pMD18T vector to form pMD18T-mE7. The pET-28a(+) and pMD18T-mE7 vectors were each digested with restriction endonucleases NdeI and HindIII. Subsequent to purification with electrophoresis in a 2% agarose gel, mE7 and pET-28a(+) were ligated with T4 DNA ligase to yield the expression plasmid pET-28a(+)-mE7. PCR, polymerase chain reaction; HPV, human papillomavirus; ori, origin of replication; mE7, mutant non-transforming HPV16 E7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356387&req=5

f1-ol-09-04-1851: Construction of the pET-28a(+)-mE7 plasmid. The HPV16 mE7 gene was amplified by splicing overlap extension PCR using pET-28a(+)-E7 as a template. The mE7 gene was then directly inserted into a pMD18T vector to form pMD18T-mE7. The pET-28a(+) and pMD18T-mE7 vectors were each digested with restriction endonucleases NdeI and HindIII. Subsequent to purification with electrophoresis in a 2% agarose gel, mE7 and pET-28a(+) were ligated with T4 DNA ligase to yield the expression plasmid pET-28a(+)-mE7. PCR, polymerase chain reaction; HPV, human papillomavirus; ori, origin of replication; mE7, mutant non-transforming HPV16 E7.
Mentions: The procedure for the construction of the pET-28a(+)-mE7 plasmid is shown in Fig. 1. The HPV16 mE7 gene was amplified by splicing PCR using pET-28a(+)-E7 as a template. The mE7 gene was then directly inserted into a pMD18T vector to form the pMD18T-mE7 plasmid. The pET-28a(+) and pMD18T-mE7 vectors were each digested with the NdeI and HindIII restriction endonucleases. Subsequent to purification by electrophoresis on a 2% agarose gel, mE7 and pET-28a(+) were ligated with T4 DNA ligase to yield the pET-28a(+)-mE7 expression plasmid. Compared with the E7 gene, mE7 was lacking nucleotides 280–294, which corresponds to residues 94–98 in the E7 protein, and nucleotide 70 in mE7 is changed from T to G, which results in residue 24 in the E7 protein being changed from Cys to Gly. The construction of the pET-28a(+)-mE7 plasmid allowed the expression of the fusion mE7 protein to have a hexa-histidine tag (his-tag) at the N-terminal end. The his-tag sequence served as a tag for affinity purification using a Ni-NTA agarose column.

Bottom Line: The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside.Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control.These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Shanghai University, Shanghai 200444, P.R. China ; Institute of Molecular Medicine, Nanjing University, Nanjing, Jiangsu 210093, P.R. China.

ABSTRACT

Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10(5) TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

No MeSH data available.


Related in: MedlinePlus