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Induction method of massive individualized natural interferon

An interferon, natural technology, applied in the fields of genetic engineering and medical biology, can solve problems such as limitations, and achieve the effects of avoiding side effects, high-efficiency anti-tumor, and anti-viral effects

Inactive Publication Date: 2011-09-14
许东升
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This also limits the clinical application of IFN [13]

Method used

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  • Induction method of massive individualized natural interferon
  • Induction method of massive individualized natural interferon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Blood Collection and Separation of Lymphocytes

[0021] Collect 10-100ml of peripheral blood from a human vein under sterile conditions, then inject the blood into a 50ml centrifuge tube, and mix it with an equal amount of phosphate-buffered saline (PBS). Add Ficoll-Pague solution to the 50ml centrifuge tube so that the ratio of blood diluted with PBS is 1:2-1:4. Then slowly add the blood diluted in PBS into the centrifuge tube so that the liquid level does not disperse. Lymphocytes were obtained by centrifugation at room temperature and at a speed of 400×g. Wash 3 times with PBS, remove the supernatant, and place the separated lymphocytes on a cell culture plate with a cell density of 10 7 / ml.

Embodiment 2

[0022] Example 2 Construction of recombinant adenovirus Ad-mtLMP1 carrying LMP1 mutant gene

[0023] (1) Construction of eukaryotic expression plasmid pDC315-LMP1

[0024] Primers were designed according to the sequence of LMP1 in GeneBank as follows:

[0025] P1: 5'-ATGGAACGCGACCTTGAGAGGGGCCCACCGGGCCC-3';

[0026] P2: 5'-GGGCCCGGTGGGCCCCCTTCAAGGTCGCGTTCCAT-3'

[0027] An EcoRI restriction site was introduced into the upstream primer, and a BamHI restriction site was introduced into the downstream primer. The total RNA of peripheral blood was extracted with Trizol one-step method, and cDNA was synthesized by reverse transcription with Oligo (dT) as a primer, and a PCR reaction system was established. PCR amplification conditions were as follows: ①pre-denaturation at 94°C for 5 min, ②denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min and 20 s for a total of 32 cycles, and ③extension at 72°C for 5 min. Take 5μl of the reaction product and ...

Embodiment 3

[0037] Example 3 Infection of peripheral blood B lymphocytes by recombinant adenovirus Ad-mtLMP1 and Sendai virus

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PUM

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Abstract

The invention belongs to the technical field of biomedicine and discloses a generation method of individualized natural interferon. Deletion mutation is carried out on a latent membrane protein 1 (LMP1) carcinogenicity segment which has 495bp length and is located between two NcoI sites at a C terminal of encoding Epstein-Barr (EB) virus LMP1, and a capability that the segment converts B lymphocyte into lymphoblastic is maintained. An LMP1 deletion mutant is inserted to an adenovirus vector for infecting peripheral blood B lymphocyte; and after being induced by sendai virus, the B lymphocyte can express the massive individualized natural interferon. The natural interferon has the functions of exciting individual immune response and inhibiting tumor and hepatitis. By using the method, the prepared interferon can efficiently exert anticancer and antiviral effects and simultaneously, side effects generated by genetic engineering interferon are avoided.

Description

technical field [0001] The invention relates to the fields of genetic engineering and medical biotechnology. Specifically, it is a mass individualized natural interferon induction method. It involves using PCR technology to obtain the LMP1 nucleotide sequence of Epstein-Barr virus, and then using overlapping PCR technology to delete and mutate the 495bp oncogenic fragment between the two Nco I sites at the C-terminus of LMP1, but maintain its transformation of B lymphocytes into lymphocytes mother cell capacity. The obtained LMP1 deletion mutant is cloned into an adenovirus vector, infected with peripheral blood lymphocytes of the patient, and then induced by Sendai virus to produce a large amount of individualized natural interferon, which can effectively exert its anti-tumor and anti-viral effects when applied to patients. At the same time, the side effects caused by genetically engineered interferon are avoided. Background of the invention [0002] Interferon (Interfer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/555C12N15/38C12N15/861A61K38/21A61P31/00A61P35/00A61P37/02
Inventor 许东升
Owner 许东升
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