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Construction and detection of the tissue-specific pINV-HPV16 E6/7 vector.

Gao H, Huang Z, Shi C, Li H - Oncol Lett (2014)

Bottom Line: The human involucrin (hINV) promoter (pINV) that contains 2474 bp of hINV upstream sequence is able to regulate tissue-specific gene expression.The carcinogenic fraction of the E6/7 gene was removed and the remaining section was cloned into T vectors, sequenced correctly and then cloned into the eukaryotic expression vector pCEP4, which was lacking the CMV promoter.The positive recombinants were identified using blue-white screening and endonuclease digestion, subsequent to sequencing and analysis, and the tissue-specific recombinant pINV-HPV16E6/7 plasmids was detected.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Clinical Medical College, Yangzhou University, Yangzhou, Jiangsu 225001, P.R. China.

ABSTRACT

A tissue-specific promoter can control downstream gene expression in tissues or organs. The human involucrin (hINV) promoter (pINV) that contains 2474 bp of hINV upstream sequence is able to regulate tissue-specific gene expression. This tissue specificity may be important for the prevention and treatment of human papilloma virus infections. pINV was cloned by polymerase chain reaction and the human papillomavirus (HPV)16 E6/7 gene was obtained from the cancer tissue samples of patients with cervical carcinoma at the Yangzhou Maternal and China Health-Care Center of Jinagsu Province (Yangzhou, China). First, specific primers were designed according to the genomic DNA sequence of the HPV16-type standard strain that has been reported and the E6/7 gene was acquired by PCR. The carcinogenic fraction of the E6/7 gene was removed and the remaining section was cloned into T vectors, sequenced correctly and then cloned into the eukaryotic expression vector pCEP4, which was lacking the CMV promoter. The positive recombinants were identified using blue-white screening and endonuclease digestion, subsequent to sequencing and analysis, and the tissue-specific recombinant pINV-HPV16E6/7 plasmids was detected.

No MeSH data available.


Related in: MedlinePlus

Identification of the pINV/HPV16E6/7 recombinant plasmid. Lane M, DL15000 marker; lane 1, digestion by Sal yielded fragments 8.9 and 3.8 kb in length; lane 2, double digestion by Not and BamH yielded fragments 9.5 and 3.2 kb in length; lane 3, double digestion of Not and Xho yielded fragments 10.2 and 2.5 kb in length; lane 4, double digestion of Xho and BamH yielded fragments 11.9 and 780bp in length. pINV, human involucrin promoter; HPV, human papillomavirus.
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f6-ol-09-02-0857: Identification of the pINV/HPV16E6/7 recombinant plasmid. Lane M, DL15000 marker; lane 1, digestion by Sal yielded fragments 8.9 and 3.8 kb in length; lane 2, double digestion by Not and BamH yielded fragments 9.5 and 3.2 kb in length; lane 3, double digestion of Not and Xho yielded fragments 10.2 and 2.5 kb in length; lane 4, double digestion of Xho and BamH yielded fragments 11.9 and 780bp in length. pINV, human involucrin promoter; HPV, human papillomavirus.

Mentions: Following the double digestion of the pGEM-T-E6/7 plasmid, the E6/7 fragments were recovered and linked with the recovered pCEP4/pINV(-PCMV). The product was then introduced into cells and the cells positive for the plasmids were selected. The plasmids were identified by single and double digestion using SalI, NotI and BamHI, and NotI and XhoI, respectively. On 1% agarose gel electrophoresis, the XhoI and BamHI double digestion yielded visible bands at 8.9, 3.8, 9.5, 3.2, 10.2, 2.5 and 11.9 kb, and 780 bp (Fig 6), which were consistent with the expected fragment sizes.


Construction and detection of the tissue-specific pINV-HPV16 E6/7 vector.

Gao H, Huang Z, Shi C, Li H - Oncol Lett (2014)

Identification of the pINV/HPV16E6/7 recombinant plasmid. Lane M, DL15000 marker; lane 1, digestion by Sal yielded fragments 8.9 and 3.8 kb in length; lane 2, double digestion by Not and BamH yielded fragments 9.5 and 3.2 kb in length; lane 3, double digestion of Not and Xho yielded fragments 10.2 and 2.5 kb in length; lane 4, double digestion of Xho and BamH yielded fragments 11.9 and 780bp in length. pINV, human involucrin promoter; HPV, human papillomavirus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4301524&req=5

f6-ol-09-02-0857: Identification of the pINV/HPV16E6/7 recombinant plasmid. Lane M, DL15000 marker; lane 1, digestion by Sal yielded fragments 8.9 and 3.8 kb in length; lane 2, double digestion by Not and BamH yielded fragments 9.5 and 3.2 kb in length; lane 3, double digestion of Not and Xho yielded fragments 10.2 and 2.5 kb in length; lane 4, double digestion of Xho and BamH yielded fragments 11.9 and 780bp in length. pINV, human involucrin promoter; HPV, human papillomavirus.
Mentions: Following the double digestion of the pGEM-T-E6/7 plasmid, the E6/7 fragments were recovered and linked with the recovered pCEP4/pINV(-PCMV). The product was then introduced into cells and the cells positive for the plasmids were selected. The plasmids were identified by single and double digestion using SalI, NotI and BamHI, and NotI and XhoI, respectively. On 1% agarose gel electrophoresis, the XhoI and BamHI double digestion yielded visible bands at 8.9, 3.8, 9.5, 3.2, 10.2, 2.5 and 11.9 kb, and 780 bp (Fig 6), which were consistent with the expected fragment sizes.

Bottom Line: The human involucrin (hINV) promoter (pINV) that contains 2474 bp of hINV upstream sequence is able to regulate tissue-specific gene expression.The carcinogenic fraction of the E6/7 gene was removed and the remaining section was cloned into T vectors, sequenced correctly and then cloned into the eukaryotic expression vector pCEP4, which was lacking the CMV promoter.The positive recombinants were identified using blue-white screening and endonuclease digestion, subsequent to sequencing and analysis, and the tissue-specific recombinant pINV-HPV16E6/7 plasmids was detected.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Clinical Medical College, Yangzhou University, Yangzhou, Jiangsu 225001, P.R. China.

ABSTRACT

A tissue-specific promoter can control downstream gene expression in tissues or organs. The human involucrin (hINV) promoter (pINV) that contains 2474 bp of hINV upstream sequence is able to regulate tissue-specific gene expression. This tissue specificity may be important for the prevention and treatment of human papilloma virus infections. pINV was cloned by polymerase chain reaction and the human papillomavirus (HPV)16 E6/7 gene was obtained from the cancer tissue samples of patients with cervical carcinoma at the Yangzhou Maternal and China Health-Care Center of Jinagsu Province (Yangzhou, China). First, specific primers were designed according to the genomic DNA sequence of the HPV16-type standard strain that has been reported and the E6/7 gene was acquired by PCR. The carcinogenic fraction of the E6/7 gene was removed and the remaining section was cloned into T vectors, sequenced correctly and then cloned into the eukaryotic expression vector pCEP4, which was lacking the CMV promoter. The positive recombinants were identified using blue-white screening and endonuclease digestion, subsequent to sequencing and analysis, and the tissue-specific recombinant pINV-HPV16E6/7 plasmids was detected.

No MeSH data available.


Related in: MedlinePlus