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Construction of HER2/neu mRNA in vitro transcription vector and use thereof

An in vitro transcription and carrier technology, applied in the fields of biology and medicine, can solve the problems of short half-life, obvious side effects, and inconvenient administration.

Active Publication Date: 2007-05-23
上海海欣生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the defects of these methods hinder their promotion and use in tumor treatment: HER2 / neu antibody, as a biological macromolecule, has disadvantages such as poor permeability, inconvenient administration, high treatment cost, and obvious side effects; peptide Vaccine-like vaccines tend to have weak immunogenicity, are limited to certain epitopes and are limited by the haplotype of individual patients, and have limited significance in the treatment or prevention of tumors; DNA vaccines generally have low expression of antigen genes, and the induced immune response Insufficient shortcomings; although mRNA vaccines can overcome the shortcomings of the above vaccines, they still have the shortcomings of easy degradation in vivo and short half-life

Method used

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  • Construction of HER2/neu mRNA in vitro transcription vector and use thereof
  • Construction of HER2/neu mRNA in vitro transcription vector and use thereof
  • Construction of HER2/neu mRNA in vitro transcription vector and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 HER2 / neu 151-2220 Cloning of cDNA fragments

[0066] 1. Reverse transcription:

[0067] The HER2 / neu positive human breast cancer cell line SK-BR-3 (purchased from ATCC, No.: HTB-30) in good growth state was collected, and the total RNA of the cells was extracted with Trizol reagent (Invitrogen Company), and oligo-dT ( Under the guidance of Takara Company), AMV reverse transcriptase (Progema Company) was used to complete.

[0068] Add 1.2 μl of total RNA, 1.0 μl of oligo-dT and 7.8 μl of RNase-free water into a 200 μl RNase-free PCR reaction tube, mix well, centrifuge, place in a water bath at 65°C for 10 minutes, and quickly cool on ice for 5 minutes, then in the above reaction Add 4 μl of 5× buffer (AMV), 1 μl of 10 mM dNTP, 1 μl of AMV and 4 μl of RNase-free water to the system, mix well, incubate at 42°C for 1 hour, and then add 80 μl of water at 72°C for 10 minutes.

[0069] The reverse transcription product can be directly used as a template to amplif...

Embodiment 2

[0083] Example 2 Construction of in vitro transcription vector pCIpA150

[0084] 1. Amplification of polyadenylic acid (polyA)

[0085] A 150bp oligonucleotide polyA was amplified by PCR method.

[0086] Template: plasmid HNA6G1 (purchased from the Institute of Immunology, Second Military Medical University).

[0087] The sequence of the amplification primer (synthesized by Shanghai Sangon Biotechnology Co., Ltd.) is:

[0088] 5'-ACAGGCCTCCACTCCGTTTGCTG-3' (SEQ ID NO: 4) and

[0089] 5'-CCTCGAGGGATACTCTAGAGCG-3' (SEQ ID NO: 5).

[0090] In a 200 μl PCR reaction tube, add: 1 μl of plasmid (concentration: 100ng / μl), 4 μl of dNTP Mixture, 0.5 μl each of primers (concentration: 10 μM), 2.5 μl of 10×Pyrobest buffer, 0.125 μl of Pyrobest Enzyme and 16.375 μl of water. After mixing, complete the reaction on a PCR machine.

[0091] Reaction parameters: 95°C: 1 minute; 94°C: 30 seconds; 58°C: 30 seconds; 72°C: 30 seconds; 25 cycles; 72°C: 10 minutes.

[0092] The reaction product...

Embodiment 3

[0101] Example 3 Construction of HER2 / neu mRNA in vitro transcription vector pHHX17

[0102] 1. Enzyme digestion of vector pCIpA150

[0103] The vector pCIpA150 was double-digested with Xho I / Kpn I (Takara Company).

[0104]In a 1.5ml PCR reaction tube, add in sequence: 5 μl of small-scale prepared plasmid, 1 μl of 10× buffer M, 0.5 μl of Hpa I Enzyme, 0.5 μl of Kpn I Enzyme and 3 μl of water. Mix well, incubate at 37°C for 1.5 hours, perform 1% agarose gel electrophoresis, and recover the target fragment from the gel.

[0105] 2. Enzyme digestion of vector pMD-HER2 / neu

[0106] Double digestion with Kpn I and Sal I.

[0107] In a 1.5ml PCR reaction tube, add in sequence: 5 μl of miniprepared plasmid, 1.5 μl of 10× buffer T, 0.5 μl of Sal I Enzyme, 0.5 μl of Kpn I Enzyme, and 2.5 μl of water. Mix well, incubate at 37°C for 1.5 hours, perform 1% agarose gel electrophoresis, incubate at 37°C for 1.5 hours, and perform 1% agarose gel electrophoresis on the digested product. ...

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Abstract

The invention provides an HER2 / neu nucleic acid molecular for producing remedial vaccine for HER2 positive tumor and its applications. It includes the following elements: (a) a 7-methyl guanine cap sequence at 5'end, (b) a 110-200bp polyA tail at 3'end, (c) the coding sequences of HER2 / neu between elements (a) and (b). The said coding sequences includes multiple CTL epitopes that have been proved, do not include its PTK active domain eliminating its PTK activity, can be used for modifying dendritic cells, induce to produce specific immune response aiming at HER2 positive tumor, and can be used for preventing and treating HER2 positive tumor such as breast carcinoma. The invention also provides the said the method for constructing HER2 / neu mRNA in vitro transcription vector.

Description

Technical field: [0001] The present invention relates to the fields of biology and medicine, and more specifically relates to the construction and use of a HER2 / neu mRNA in vitro transcription carrier and its application in the prevention and treatment of HER2 / neu positive tumors. technical background: [0002] HER2 / neu is an oncogene that often changes in human tumors and is closely related to the occurrence and development of tumors. It is the second member of the epidermal growth factor receptor (EGFR) family. The epidermal growth factor receptor family, also known as HER or ErbB2 family, plays an important role in cell signal transduction and is an important regulator of cell growth, differentiation and survival. The human HER2 / neu gene is located on the short arm of chromosome 17, and the expression product is a single-chain transmembrane glycoprotein with a molecular weight of 185KDa, namely p185. [0003] p185 contains 1255 amino acid residues, the intracellular segm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63A61K48/00A61K35/12A61K39/00A61P35/00A61K35/15
Inventor 万涛王燕兰王建莉
Owner 上海海欣生物技术有限公司
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