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AFP and GM-CSF dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector

A technology of GM-CSF and recombinant vector, which is applied in the field of recombinant vector and its preparation, can solve the problems of lack of immunotherapy targeting, immune regulation, and small anti-tumor ability, achieve good liver cancer immunotherapy effect, and promote T cell Immune response, effect of dose reduction

Active Publication Date: 2015-06-24
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current GM-CSF gene immunotherapy method only locally increases the expression level of GM-CSF protein in the body, its immune regulation effect and anti-tumor ability are small, and it lacks the targeting of immunotherapy

Method used

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  • AFP and GM-CSF dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector
  • AFP and GM-CSF dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector
  • AFP and GM-CSF dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Obtaining AFP gene fragments containing specific enzyme cleavage sites

[0047] 1. Primer design

[0048] According to the nucleotide sequence of the AFP gene (as shown in SEQ ID NO: 1 in the sequence listing) and the expected insertion multiple cloning site on the pIRES2-EGFP plasmid vector, design specific primers as follows:

[0049] AFP upstream primer (as shown in SEQ ID NO:4 in the sequence listing):

[0050] 5'-GC AGATCT ATGAAGTGGGTGGAA-3' (the underlined part is the sequence of the Bgl II restriction site),

[0051] AFP downstream primer (as shown in SEQ ID NO:5 in the sequence listing):

[0052] 5'-TT GAATTC TTAAACTCCCAAAAGCAGC-3' (the underlined part is the sequence of EcoR I restriction site).

[0053] 2. Obtain cDNA template

[0054] RNA was extracted from human liver cancer cell HepG2 by TRIzon method (TRIzon total RNA extraction kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number is CW0580), and reverse...

Embodiment 2

[0061] Example 2: Construction of pIRES2-AFP-EGFP recombinant vector

[0062] Using restriction endonucleases Bgl II and EcoR I, digest the pIRES2-EGFP plasmid (the multiple cloning site of the plasmid contains Bgl II, EcoR I restriction sites) and the AFP gene fragment obtained in Example 1, respectively, to obtain Linearized pIRES2-EGFP vector and digested AFP gene sequence after enzyme digestion; use T4 DNA ligase system for ligation reaction, incubate at 22°C for 30 minutes, and then inactivate at 70°C for 5 minutes to construct pIRES2-AFP -EGFP recombinant vector (such as image 3 shown).

[0063] Structural features of the pIRES2-EGFP plasmid (eg figure 2 Shown) It can be seen that after the AFP gene is inserted into the multiple cloning site of the pIRES2-EGFP plasmid, it is located upstream of the self-sequence IRES of the plasmid vector (such as image 3 shown), that is, the AFP and EGFP sequences under the same promoter were expressed separately.

[0064] 1. Dou...

Embodiment 3

[0090] Example 3: Double PCR method to obtain GM-CSF gene fragments with restriction endonuclease cohesive ends

[0091] According to the GM-CSF gene sequence and the expected insertion multiple cloning site on the pIRES2-EGFP plasmid vector, design two pairs of primers with different lengths and sticky ends with restriction endonucleases; reverse transcribe with RNA extracted from CIK cells The cDNA of cDNA was used as a template, and the above two pairs of primers were used for PCR amplification to obtain two PCR amplification products; the two PCR amplification products were mixed and then denatured and annealed sequentially to obtain four GM-CSF gene fragments, wherein Both GM-CSF gene fragments have restriction endonuclease cohesive ends, which allow directional ligation of the GM-CSF gene fragments into the desired polyclonal of the plasmid vector without the need for restriction endonuclease digestion site.

[0092] Compared with the traditional PCR product cloning met...

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Abstract

The invention discloses an AFP and GM-CSF dual-gene co-expression recombinant vector, which is sequentially linked with an AFP gene, an IRES sequence and a GM-CSF gene in a vector transcription direction, or is sequentially linked with a GM-CSF gene, an IRES sequence and an AFP gene in a vector transcription direction, wherein nucleotide sequence of the AFP gene is represented in SEQ ID NO: 1 in a sequence list, nucleotide sequence of the GM-CSF gene is represented in SEQ ID NO: 2 in the sequence list, and nucleotide sequence of the IRES sequence is represented in SEQ ID NO: 3 in the sequence list. The dual-gene co-expression recombinant vector, which links the AFP gene and the GM-CSF gene through the IRES sequence, can simultaneously express alpha fetal protein and a granulocyte-macrophage colony stimulating factor in a same vector; and the recombinant vector, in immunogene therapy of liver cancer, not only can develop an immune regulating function of a cell factor but also can generate a specific anti-tumor effect targeted for the liver cancer.

Description

technical field [0001] The invention relates to a recombinant vector and its preparation method and application, in particular to a double-gene co-expression recombinant vector and its preparation method and application. Background technique [0002] Primary hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is one of the most common malignant tumors. my country is a high-incidence area of ​​liver cancer. The annual incidence of liver cancer accounts for more than 50% of the world, and the mortality rate of liver cancer has accounted for the national tumor mortality rate. The second is a disease that seriously threatens people's lives and health. Surgical resection is currently the most curative treatment for liver cancer, and the technology of liver transplantation is quite mature. However, due to the high cost and difficulty in donor sources, the use of anti-rejection drugs after surgery will lead to faster growth and metastasis of residual cancer due to immunosuppres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66A61K48/00A61P35/00A61P1/16
Inventor 栗炳南丰慧根左百乐林俊堂曹毓林
Owner XINXIANG MEDICAL UNIV
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