Expression sequence and application of a liver cancer targeting dual group apoptosis protein

An apoptotic protein and liver cancer technology, which is applied in the field of preparation of targeted tumor gene therapy drugs, can solve problems in research, production technology, production process, application development and other problems

Inactive Publication Date: 2011-12-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the third-generation adenovirus has obvious advantages, its production process is a major factor that hinders its application development. There are still problems in production technology, and it is still only in the research stage.

Method used

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  • Expression sequence and application of a liver cancer targeting dual group apoptosis protein
  • Expression sequence and application of a liver cancer targeting dual group apoptosis protein
  • Expression sequence and application of a liver cancer targeting dual group apoptosis protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Cloning of human alpha-fetoprotein enhancer and promoter and construction of transcriptional regulatory elements

[0016] Genomic DNA was extracted from fresh normal human blood, and the human alpha-fetoprotein enhancer and promoter were amplified by using characteristic primers to amplify the human AFP enhancer and promoter using the polymerase chain reaction (PCR) technology, thereby constructing the AFP enhancer and promoter transcriptional regulatory elements.

[0017] The upstream and downstream primer sequences of the AFP enhancer are:

[0018] Seq No.2 5'-AGA TCT CAG ATT GAA TTA TTT GCC TGT CA-3'

[0019] Seq No.3 5'-GGA TCC TAG GAA GTT TTC GCA ATA ATA C-3'

[0020] The sequences of the upstream and downstream primers for the AFP promoter are:

[0021] Seq No.4 5'-GGA TCC AAA TCA TGC TGA AAT TCT TTT ATA CTC-3'

[0022] Seq No.5 5'-AGA TCT GCC CCA AAG AGC TCT GTG T-3'.

[0023] Use Seq No.2 and Seq No.3 as primers to amplify the AFP enhancer from the...

Embodiment 2

[0025] Example 2: Construction of recombinant activated Caspase-3 gene, cloning of Granzyme B gene and construction of adenovirus shuttle plasmid (see attachment for details of primer sequences)

[0026] Using P1 (Seq No.6) and P2 (Seq No.7) and P1 (Seq No.6) and P7 (Seq No.12) as primers, from pcDNA4 / HisMax Amplify the SP163 sequence in A vector, which can enhance the protein expression level of the regulated gene; use P3 (SEQ NO.8) and P4 (SEQ NO.9) as primers to amplify the small Caspase-3 from human genomic DNA Subunit unit; using P5 (SEQ NO.10) and P6 (SEQ NO.11) as primers, amplifying the large subunit unit of Caspase-3 from human genomic DNA; using P8 (SEQ NO.13) and P9 (SEQ NO.13) NO.14) is a primer for amplifying the Granzyme B gene sequence from human genomic DNA. Through the ligation reaction, the amplified gene fragment is connected with the AFP transcriptional regulatory element and the Poly A tail to construct a completed expression cassette. For the construct...

Embodiment 3

[0036] Example 3 Killing effect of adenovirus Ad5-reCASP3GB, Ad5-reCASP3 and Ad5-GB on liver cancer cells and non-liver cancer cells

[0037] Liver cancer cells Hep3B, HepG2, SMMC7721 and non-liver cancer cells Bcap37 in the logarithmic growth phase were taken, according to 2×10 3 The density of cells / well was seeded in 96-well plates and cultured for 24 hours to allow them to adhere to the wall. Dilute adenovirus with culture medium to appropriate concentration, add Ad5-reCASP3GB, Ad5-reCASP3 and Ad5-GB and Ad5-GFP at MOI of 200pfu / cell, 100pfu / cell, 50pfu / cell, 10pfu / cell, 1pfu / cell and Opfu / cell infects cells. After 4 days of virus infection, 20 ul of MTT solution (5 mg / ml) was added to each well, the culture was continued at 37° C. for 4 hr, and the culture was terminated. Discard the culture supernatant, add 200ul of DMSO to each well, and shake for 10min to fully dissolve the crystals. Select a wavelength of 570nm, and measure the OD value of each well on an enzyme-l...

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Abstract

The present invention provides a liver cancer targeting dual-group apoptotic protein expression sequence, which is a DNA sequence in which the specific AFP promoter regulates the expression of two apoptotic proteins, Caspase 3 and Granzyme B, and has the nucleoside shown in SEQID No: 1 acid sequence. The present invention utilizes the targeted regulatory effect of the AFP enhancer / promoter transcriptional regulatory element, so that the exogenous gene is only expressed in the AFP-positive liver cancer cells, so as to achieve a targeted and efficient anti-tumor effect; a recombinant activated Caspase- 3 molecule, and apply it to promote apoptosis of target cells; introduce granzyme B, and fuse granzyme B with recombinant activated Caspase-3 to mediate apoptosis of target cells. It can be used in the preparation of targeted gene medicine for treating AFP-positive liver cancer.

Description

technical field [0001] The present invention belongs to biological technology, and relates to the regulation of gene expression, in particular, to the design and preparation of DNA expressing a recombinant apoptosis-promoting protein specific for alpha-fetoprotein-positive liver cancer and its application in alpha-fetoprotein-positive liver cancer cells. Specifically expressed, its product can effectively induce the apoptosis of liver cancer cells. The invention can be used to prepare targeted tumor gene therapy drugs. Background technique [0002] Replication-defective adenovirus is the most classic form of adenovirus vector, it is a kind of adenovirus that cannot proliferate in cells, it only acts as a transport carrier, and can carry foreign genes into cells. The expression products of exogenous genes affect the growth of cells, thus playing the role of gene therapy. According to the mechanism of action of replication-defective adenovirus, in order to improve its therap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11A61K48/00A61P1/16A61P35/00
Inventor 曹江毛晨宇陈萍王浩浩陈喆滕理送
Owner ZHEJIANG UNIV
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