Nucleotide sequence and application thereof

A nucleotide sequence, Epstein-Barr virus technology, applied in nucleic acid vectors, medical preparations containing active ingredients, introduction of foreign genetic material using vectors, etc., can solve the problem of no Epstein-Barr virus subunit vaccine report and other problems

Inactive Publication Date: 2016-11-09
JINING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the potential carcinogenicity of EBV, live attenuated EBV vaccines are not suitable for the prevention of healthy people. Vaccine research based on EBV subunit vaccines has become a hot research topic at home and abroad, but so far there is no report of EBV subunit vaccines

Method used

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  • Nucleotide sequence and application thereof
  • Nucleotide sequence and application thereof
  • Nucleotide sequence and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The synthetic process of embodiment 1 nucleotide sequence SEQ ID No.1

[0021] The nucleotide sequence of SEQ ID No.1 is composed of the cDNA sequence of IL-2, the cDNA sequence of LMP2A and the fusion fragment. The nucleotide sequence of the cDNA sequence of IL-2 is shown in SEQ ID No.2. The cDNA of LMP2A The nucleotide sequence of the sequence is shown in SEQ ID No.3, and the nucleotide sequence of the fusion fragment is shown in SEQ ID No.4. The specific synthesis process of the nucleotide sequence of the present invention is as follows:

[0022] Using IL-2 cDNA as a template, upstream primer F1, downstream overlapping primer F2, PCR amplification, pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 45 seconds, extension at 72°C for 2 minutes; denaturation-annealing-extension process 60 cycles were performed, with a final extension of 10 min at 72°C.

[0023] The IL-2 fragment is obtained, the nucleotide sequence of F1...

Embodiment 2

[0026] Embodiment 2 Connection and transformation of fusion gene LMPI-2 and plasmid pMV261

[0027] After the purified gene LMPI-2 and plasmid pMV261 were digested with EcoR I and Sal I, they were ligated; 0.1-0.3 pmoL of LMPI-2 gene fragment, 1 μL of pMV261, T 4 DNA ligase 1.5 μL with ddH 2 O was adjusted to 5 μL, then 5 μL of ligation buffer was added, and reacted overnight at 16°C.

[0028] Apply 50 μl of isopropylthiogalactopyranoside (IPTG) on the prepared bacterial basal medium LB solid plate, and place it at room temperature for 2 hours. Take out the competent E.coli DH5α cells from the ultra-low temperature refrigerator and thaw in the ice bath. Take 30-40 μl of E.coliDH5α cells and gently mix the recombinant plasmid, ice-bath for 20-30 minutes, heat shock at 42°C for 90 seconds, immediately ice-bath for 3 minutes; add 1ml of liquid LB medium (without antibiotics), and incubate at 37°C for 2 hours; 12 Centrifuge at 000r / min (radius 10cm) for 3min, and redissolve the...

Embodiment 3

[0029] Construction and expression of embodiment 3 recombinant BCG

[0030] Preparation of Bacillus Calmette-Guérin (BCG) Competent: BCG (Bacillus Calmette-Guérin, referred to as BCG, Chinese name comes from its inventor Karl-Jie) is a vaccine for the prevention of tuberculosis, using live avirulent Mycobacterium bovis (Mycobacterium bovis) made.

[0031]BCG was cultured in Middlebrook7H9 medium at 37°C on a shaker (rotating speed: 150rpm); when the OD600 value of the culture solution was about 0.6, add the BCG bacterial liquid into the centrifuge tube and put it in an ice bath for 2 hours; Pre-cool for 2 hours; centrifuge at 4°C for 15 minutes at 8,000 rpm, discard the supernatant; resuspend the bacteria with 1 / 10 of the original volume and pre-cooled 10% glycerol; repeat the above operation for a total of five times; discard the supernatant, 1 / 50 of the original volume was added to resuspend the bacterial cells in 10% glycerol to obtain competent bacteria, which were placed...

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Abstract

The invention relates to a nucleotide sequence and application thereof and particularly relates to a nucleotide sequence which is obtained after fusion and optimization of a cDNA sequence of 1L-2 and a cDNA sequence of LMP2A. A live vaccine is obtained; with virogenes and gene products as targets, cancer cells are killed selectively by utilizing the difference between epstein-barr virus (EBV) positive tumor cells and normal cells, and a novel method and the experimental basis are provided for specific treatment of virus-associated tumors.

Description

technical field [0001] The invention relates to a nucleotide sequence and its use, belonging to the field of molecular biology technology. Background technique [0002] Epstein-Barr virus (EBV) is a human lymphocyte-tropic γ-herpes virus discovered in tumor cell culture by Epstein and Barr in 1964 when they were studying malignant lymphoma in African children. The full-length genome is 184kb. There are currently two subtypes of EBV that can infect humans: EBV-1 and EBV-2, the difference lies in the genetic makeup of the encoding nuclear antigen. EBV can cause two different types of infections, one is proliferative infection and the other is non-proliferative infection. Under certain conditions or under the action of certain inducing factors, the latent EBV genome can be activated and transformed into proliferative infection. Infect. In addition, if host cells infected and transformed by EBV are stimulated by certain auxiliary factors during the continuous division and prol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/74C12N15/11A61K39/00A61P35/00
CPCA61K39/0011A61K2039/53A61K2039/55533C07K14/4748C07K14/55C07K2319/00C12N15/11C12N15/74C12N2800/101
Inventor 薛庆节闫迎春李运清吕厚东陈廷
Owner JINING MEDICAL UNIV
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