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p66shc recombinant adenovirus vector as well as construction and application thereof

A technology of recombinant adenovirus and p66shc, which is applied in the fields of application, recombinant DNA technology, and the introduction of foreign genetic material by using vectors, and can solve the problems that the p66Shc gene has not been reported.

Inactive Publication Date: 2014-10-01
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to these reports, it is believed that the p66Shc gene is closely related to the level of reactive oxygen species in cells, but the effect of p66Shc gene on cell proliferation has not been reported yet.

Method used

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  • p66shc recombinant adenovirus vector as well as construction and application thereof
  • p66shc recombinant adenovirus vector as well as construction and application thereof
  • p66shc recombinant adenovirus vector as well as construction and application thereof

Examples

Experimental program
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Effect test

Embodiment 1p66

[0032] Construction and expression of embodiment 1p66Shc recombinant adenovirus vector

[0033] (1) Cloning of pAdenoX-p66Shc

[0034] The pAdenoX-p66Shc recombinant plasmid was digested with Xho I, and electrophoresis analysis contained five bands at 1445, 2466, 3320, 5629, and 8046. The size of the bands was consistent with the number and distribution of restriction sites on the recombinant plasmid. After further sequencing verification, it was confirmed that the recombination cloning site of pAdenoX-CMV had been accurately connected with the open reading frame of the target gene p66Shc.

[0035] (2) Production of human p66Shc adenovirus particles

[0036] The characteristic cytopathic effect (CPE) can be observed under an inverted microscope on the 10th day after transfection of the pAdeno X-p66Shc recombinant plasmid linearized by Pac I digestion into HEK293A cells, that is, the cells become round, the refraction is enhanced, and Obvious plaques and even cell shedding be...

Embodiment 2p66

[0039] Example 2 p66Shc recombinant adenovirus vector inhibits tumor cell growth

[0040] The p66Shc recombinant adenovirus vector constructed by the invention infects human cervical cancer cells (Hela cells) and exerts the effect of inhibiting the proliferation of Hela cells.

[0041] (1) The proliferation rate of Hela cells decreased significantly after being infected with p66Shc recombinant adenovirus: From the growth curve of Hela cells, it can be seen that after 24 hours of infection with p66Shc recombinant adenovirus, the proliferation rate of Hela cells continued to decrease, which was different from that of the control group (infected with green fluorescent Protein recombinant adenovirus vector (Ad-GFP) Hela cells as a negative control) was statistically significant (2nd, 3rd day, p image 3 ). The MTT experiment of Hela cells infected with human p66Shc-deficient recombinant adenovirus for 48 hours showed that when the amount of cell lysate containing p66Shc-deficient r...

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Abstract

The invention provides a p66Shc recombinant adenovirus vector. Homologous recombination in vitro of a human p66Shc fragment and a pAdeno X-CMV vector is realized by use of the gene engineering technology, and then competent bacteria are used for transformation to screen out correct recombinant adenoviruses; next, the recombinant adenoviruses are linearized and then used for transfecting HEK293A cells, and therefore, the recombinant adenoviruses are packaged and amplified largely in the HEK293A cells. After Hela cells are infected with the p66Shc recombinant adenovirus vectors, the growth and proliferation of the cells can be obviously inhibited; if human umbilical vein endothelial cells (HUVEC) are in infected with the p66Shc recombinant adenovirus vectors, no obvious growth inhibition effect is caused. The p66Shc recombinant adenovirus vector is expected to achieve a special growth inhibition effect on tumor cells.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and relates to the construction and application of p66Shc recombinant adenovirus. It uses molecular cloning technology, including PCR amplification gene, in vitro recombination connection, etc., to obtain p66Shc recombinant adenovirus vector capable of infecting various cells. The different effects of the carrier on the proliferation of human cervical cancer cells (Hela cell line) and human umbilical vein endothelial cells laid an experimental foundation for the further development of adenovirus gene therapy agents that can specifically inhibit the growth of tumor cells. Background technique [0002] p66Shc is a key protein that regulates oxidative stress and life cycle signal transduction pathways in mammalian cells, encoded by the proto-oncogene ShcA (Biochim Biophys Acta, 1797, 952-960 (2010)). The p66Shc protein contains several basic functional areas: 1) N-terminal phosphorylated tyrosin...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66A61K48/00A61P35/00
Inventor 胡刚林雅军徐蓉魏洁程建华
Owner BEIJING HOSPITAL
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