AFP (Alpha Fetoprotein) and IL-2 (Interleukin-2) double-gene co-expressing recombinant vector as well as preparation method and application thereof

A recombinant carrier and IL-2 technology, which is applied in gene therapy, recombinant DNA technology, and the use of vectors to introduce foreign genetic material, etc., can solve the problems of immune regulation, low anti-tumor ability, lack of targeting of immunotherapy, etc., to achieve Enhance the expansion ability and cell viability, improve the effect of liver cancer immunotherapy, and enhance the effect of the body's immune regulation ability

Inactive Publication Date: 2014-03-05
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current IL-2 gene immunotherapy method only locally increases the expression level of IL-2 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 (Alpha Fetoprotein) and IL-2 (Interleukin-2) double-gene co-expressing recombinant vector as well as preparation method and application thereof
  • AFP (Alpha Fetoprotein) and IL-2 (Interleukin-2) double-gene co-expressing recombinant vector as well as preparation method and application thereof
  • AFP (Alpha Fetoprotein) and IL-2 (Interleukin-2) double-gene co-expressing recombinant vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Obtaining AFP gene fragments containing specific enzyme cleavage sites

[0048] 1. Primer design

[0049] 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:

[0050] AFP upstream primer (as shown in SEQ ID NO:4 in the sequence listing):

[0051] 5'-GC AGATCT ATGAAGTGGGTGGAA-3' (the underlined part is the sequence of the Bgl II restriction site),

[0052] AFP downstream primer (as shown in SEQ ID NO:5 in the sequence listing):

[0053] 5'-TT GAATTC TTAAACTCCCAAAAGCAGC-3' (the underlined part is the sequence of EcoR I restriction site).

[0054] 2. Obtain cDNA template

[0055] 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

[0062] Example 2: Construction of pIRES2-AFP-EGFP recombinant vector

[0063] 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).

[0064] 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.

[0065] 1. Dou...

Embodiment 3

[0092] Example 3: Double PCR method to obtain IL-2 gene fragments with restriction endonuclease cohesive ends

[0093] According to the IL-2 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 the above-mentioned two pairs of primers was used as a template 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 IL-2 gene fragments, wherein Both IL-2 gene fragments have restriction endonuclease cohesive ends that allow for directional ligation of IL-2 gene fragments into desired polyclones of plasmid vectors without the need for restriction endonuclease digestion site.

[0094] Compared with the traditional PCR product cloning method, this method (double PCR ...

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Abstract

The invention discloses an AFP (Alpha Fetoprotein) and IL-2 (Interleukin-2) double-gene co-expressing recombinant vector, wherein an AFP gene, an IRES (Internal Ribosome Entry Site) sequence and an IL-2 gene are sequentially connected along a transcription direction of the vector, or the IL-2 gene, the IRES sequence and the AFP gene are sequentially connected along the transcription direction of the vector; the nucleotide sequence of the AFP gene is shown as SEQ ID NO:1 in a sequence table, the nucleotide sequence of the IL-2 gene is shown as SEQ ID NO:2 in the sequence table, and the nucleotide sequence of the IRES sequence is shown as SEQ ID NO:3 in the sequence table. According to the double-gene co-expressing recombinant vector disclosed by the invention, the AFP gene and the IL-2 gene are connected via the IRES sequence, so that the AFP gene and the IL-2 gene can be simultaneously expressed in the same vector; the recombinant vector can be applied to the genetic immunization treatment of liver cancer, thereby achieving an immunomodulatory effect of cell factors and producing a specific anti-tumor effect to liver cancer in a targeted manner.

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 Applications(China)
IPC IPC(8): C12N15/85C12N15/66A61K48/00A61P35/00A61P1/16
Inventor 曹毓琳左百乐栗炳南连杰林俊堂丰慧根
Owner HENAN HUALONG BIOLOGICAL TECH
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