Acquisition and use of tumor-selective replicative adenovirus - thymidine kinase gene construct

A selective and replicative technology, applied in virus/bacteriophage, gene therapy, antineoplastic drugs, etc., can solve the problems of unstable activity of exogenous promoters and weak efficacy of lysing tumor cells

Inactive Publication Date: 2011-10-05
周剑峰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, they still have the following disadvantages: Although the vectors designed based on the biological basis of adenoviruses have the therapeutic advantage of replicability in tumor conditions, due to various design deficiencies, the obtained adenoviruses cannot replicate in tumor cells and lyse tumors. The potency of cells is much weaker than that of wild-type adenovirus; The activity of exogenous promoters combined with current adenovirus gene therapy vectors is unstable or low in vivo

Method used

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  • Acquisition and use of tumor-selective replicative adenovirus - thymidine kinase gene construct
  • Acquisition and use of tumor-selective replicative adenovirus - thymidine kinase gene construct
  • Acquisition and use of tumor-selective replicative adenovirus - thymidine kinase gene construct

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0017] Example 1. Construction of pXC1 series mutants (Δ920-946pXC1)

[0018] pXC1 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-03). This plasmid contains the human adenovirus type 5 (Ad5) 22-5790nt sequence.

[0019] Deletion of 920-946nt by 3 times PCR method, acquisition of fragment 1: primer 1=5′-CG GGA TCC GGG CCC CCATTT CC-3', equivalent to 9883-9902nt, the underlined part is the BamHI restriction site; primer 2 = 5'- GTC ACT GGGTGG ATC GAT CAC CTC CGG TAC-3', equivalent to 922-905nt, the underlined part is complementary to primer 3; use pXC1 as template, perform PCR reaction, the total volume of the reaction system is 100 μl, including:

[0020] Contains MgCl 2 10 μl of 10x PCR buffer

[0021] 2mM dNTP 10μl

[0022] 10 μM Primer 1 1 μl

[0023] 10 μM Primer 2 1 μl

[0024] pXC1 10ng / μl 1μl

[0025] pfu high fidelity Taq enzyme 2.5u

[0026] Add water to 100μl;

[0027] The reaction conditions were: 95°C for 30...

example 2

[0033] Example 2. Construction of Δ920-946Ad5 recombinant adenovirus

[0034] pBHGE3 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-12), this plasmid contains the entire genome sequence except the Ad5 packaging signal (194-358nt).

[0035] When pBHGE3 is obtained from Microbix Biosystem Inc., the total amount is 10 μg. First, electroporate into competent bacteria, pick positive clones, and extract plasmids. The obtained plasmids are treated with CsCl 2 -EB purified by ultracentrifugation.

[0036] Homologous recombination method to obtain Δ920-946Ad5 recombinant adenovirus construct, the method is as follows:

[0037] Plant 7.5×10 in a 15cm petri dish 5 293 cells, the culture medium is 10% FBS DMEM, by the next day, the cells should be 1-1.5×10 6 , about 70% of the cells are confluent; 3-4 hours before transfection, replace with fresh culture medium.

[0038] Prepare co-transfection DNA-calcium phosphate solution: 1600 μl steri...

example 3

[0053] Example 3. Construction of the subcloning vector pCDNA3.1-Δ6.7k / gp19k in the Ad5E3 region

[0054] The Ad5E3 region is called the Ad5 early region 3. Driven by the endogenous promoter, this region encodes 7 proteins. For the sequence, structure and function, please refer to the appendix figure 1 : 12.5k, 27858-28179nt, function unknown; 6.7k, 27547-28736nt, together with RID complex, inhibits the expression of TRAIL receptor 1 and 2 on the cell surface; gp19k, 28735-29215nt, binds to MHC class I antigen, inhibits its Presented to the cell surface, evading the clearance of CTL; ADP, 29419-29770nt, lyses cells and releases virus; RIDα, 29784-30057nt, forms a complex with RIDβ, prevents the lysis of TNF, and clears FAS antigen; RIDβ, 30062-30458nt and 14.7k, 30453-30837, inhibits the cleavage of TNF.

[0055] The purpose of this experiment is to delete the 28530-29360nt region of the E3 region and insert an exogenous therapeutic gene into the E36.7k / gp19k region of the re...

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PUM

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Abstract

Belonging to the genetic engineering field in medicine, the invention discloses a construction scheme for artificially modified human adenovirus type 5 (Ad5), and specific application of a tumor-selective replicative adenovirus construct in tumor treatment. With the techniques of PCR (polymerase chain reaction) amplifying fixed point deletion, enzyme cutting, connection, cloning, homologous recombination, transfection, single cloning purification of adenovirus and the like, a recombinant adenovirus construct can be acquired. Acquisition of the construct is characterized in that: 27 bases are deleted in 2(CR2) region of E1A conserved sequence of Ad5 genome; 28530-29360nt in 6.7k/gp19k gene in E3 region are deleted, and meanwhile a fragment of full-length cDNA(1131bp) of HSV-TK (herpes simplex virus thymidine kinase) is inserted into the deletion region. The construct of the invention is a novel oncolytic adenovirus carrier with high tumor-selective replication capability. With the suicide gene of HSV-TK and dissolubility of tumor cells by oncolytic viruses, the dual killing effects have unique utility value in tumor biotherapy.

Description

1. Technical field [0001] The present invention relates to a human adenovirus type 5 (Human adenovirus type 5, Ad5) recombinant construction scheme, which is characterized in that: the 920-946 nt of the Ad5 genome is directional deleted, that is, GAT CTT ACC TGC CAC GAG GCTGGC TTT, the The sequence encodes amino acids 121 to 129 of the E1A protein. On this basis, the 28530-29360 nt of the Ad5E3 region is further deleted, and a ClaI restriction site is introduced into the above deletion region; at the same time, the full-length coding sequence of the HSV-TK gene is inserted into the deletion region (1131bp, see the nucleic acid sequence list for the specific sequence), so as to obtain the tumor-selective replication-type adenovirus construct Ad5 / ΔE1A / Δ6.7k / gp19k / HSV-TK that has a therapeutic effect on tumors. The content of the invention belongs to the technical field of medical genetic engineering. 2. Background technology [0002] At present, malignant tumors are one of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/54C12N15/63A61K48/00A61P35/00
Inventor 周剑峰马丁韩志强卢运萍王世宣
Owner 周剑峰
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