Recombinant human esoderma colyone adenovirus, and its preparing method and use

A technology of endostatin and adenovirus, which is applied in the field of genetic engineering, can solve the problems of complex and time-consuming protein purification process, large clinical application restrictions, and high cost, and achieve inhibition of growth and migration, inhibition of growth and metastasis, and increased administration time The effect of the interval

Active Publication Date: 2009-06-03
GUIZHOU BAILING GRP PARMACEUTIAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, it is believed that angiogenesis inhibitors, a large class of proteins, have many limitations in treatment: First, due to their pharmacokinetic characteristics, the protein molecules expressed in vitro are unstable, which determines that protein infusion is a long-term, repeated, and sufficient treatment. The large dosage and short-term multiple infusions have brought great inconvenience to patients; in addition, the protein purification process is complicated, time-consuming, low yield, high cost, and relatively limited clinical application

Method used

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  • Recombinant human esoderma colyone adenovirus, and its preparing method and use
  • Recombinant human esoderma colyone adenovirus, and its preparing method and use
  • Recombinant human esoderma colyone adenovirus, and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Preparation of human endostatin gene

[0061] In this example, the RT-PCR method was used to amplify the cDNA fragment of human endostatin from the total RNA of human liver.

[0062] Design and synthesize RT-PCR primers according to the human endostatin gene sequence, in order to directly insert the PCR product into the cloning vector—pUC18 (purchased from Invitrogen Company), the BamH I restriction site was designed in both the 5' primer and the 3' primer point, the primer sequences are as follows:

[0063] 5' Primer: 5'CGG GAT CCA CAG CCA CCG CGA CTT CCA GCC 3'

[0064] 3' Primer: 5'GCG GAT CCTACT TGG AGG CAG TCA TGA AGC 3'

[0065] RT-PCR conditions are as follows:

[0066] After the RT-PCR reaction mixture was denatured at 50°C for 30 minutes, the reaction was carried out according to the following conditions:

[0067] Reverse transcription reaction: denaturation at 94°C for 30 seconds; annealing at 50°C for 30 seconds; extension at 68°C for 1 minute. ...

Embodiment 2

[0070] Example 2 Preparation of human endostatin gene encoding human IL-2 gene signal peptide

[0071] With reference to the cDNA sequence of human IL-2 (GI: 28178860), PCR primers were designed and synthesized as follows:

[0072] 5’ Primer: 5’CGG ATC CAT GTA CAG GAT GCA ACT CCT GTC TTG CAT TGCACT AAG TCT TGC ACT TGT CAC GAA TTC GGC CCA CAG CCA CCG CGA CTTCCA GCC 3’

[0073] 3’ Primer: 5’GCG GAT CCT ACT TGG AGG CAG TCA TGA AGC 3’

[0074] Both the 5' primer and the 3' primer were designed with a BamH I restriction site, and were directly inserted into the shuttle vector pAdenovator-CMV5; the italic part of the 5' primer was the signal peptide sequence of human IL-2.

[0075] Then use pUC18-endo as a template to amplify the human endostatin DNA fragment containing the IL-2 gene signal peptide, and the PCR conditions are as follows:

[0076] Denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds; extension at 72°C for 30 seconds. React for 30 cycles. This was...

Embodiment 3

[0078] Example 3 Construction and screening of recombinant adenovirus

[0079] 1. After constructing the cloned human endostatin gene containing human IL-2 signal peptide into the pAdenoVator-CMV5 shuttle vector, it was combined with the circular plasmid pAdenoVatorΔE1E3 (purchased from Qbiogene Corporation) containing the adenovirus genome (E1, E3 deletion) ) co-transform Escherichia coli BJ5183 (purchased from Qbiogene Company) by conventional electroporation method, and screen recombinant pAd-endo for identification. The results show that the recombinant pAd-endo (1) and pAd-endo (3) with two kinds of recombination sites have been screened respectively (see image 3 ). The homologous recombination site is located in the 3.0 kb cut out fragment of the left arm, and the recombination site is located in the 4.5 kb cut out fragment of the replicon.

[0080] 2. The linearized recombinant plasmid pAd-endo and liposome Lipofectamine (purchased from Invitrogen) were co-transfecte...

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Abstract

The invention supplies a new recombination endostatin gene that connects the 3' end of IL-2 gene signal peptide sequence with the 5' end of endostatin coding sequence, and the gene could be constructed into adenovirus and make anti-tumor injection. The injection would restrain the growth and transferring of tumor in body and keep entire bioactivity. It has the advantages of lowering medicine cost, improving life quality of patients and good market prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an adenovirus containing recombinant human endostatin gene and its preparation method and application. Background technique [0002] In 1971, Folkman first proposed that tumor growth and metastasis are dependent on blood vessels. Studies in recent decades have continuously confirmed this point of view. The occurrence, development and metastasis of tumors are closely related to tumor angiogenesis, which is an important control point for tumor growth and metastasis (Skobe et al., 1997; Dameron et al. ., 1994; Volpert et al., 1997; Bertolini et al., 2000; Holmgren et al., 1995). Anti-angiogenesis therapy of tumor has become a new research direction of tumor therapy. [0003] Endostatin (endostatin) is the most famous endogenous angiogenesis inhibitory factor discovered by O'Reilly et al. in 1997. It is a C-terminal fragment of collagen XVIII (endostatin protein derived from huma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/62C07K14/435C12N15/63C12N15/86C12N15/79A61K48/00A61P35/00C12N15/10A61K38/17
Inventor 魏于全杨莉
Owner GUIZHOU BAILING GRP PARMACEUTIAL CO LTD
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