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Combination immunizing preparation comprising recombinant plasmid and duplicate deficit type recombinant adenovirus

A replication-deficient, recombinant adenovirus technology, applied in the field of combined immunization preparations, which can solve problems such as non-specific damage, wandering, and treatment failure

Active Publication Date: 2007-03-14
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, radiotherapy has huge non-specific damage to the body. About 40% to 50% of patients with complete remission after treatment still have treatment failure due to distant metastasis and local recurrence, and the five-year survival rate of NPC is still hovering at 30%.

Method used

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  • Combination immunizing preparation comprising recombinant plasmid and duplicate deficit type recombinant adenovirus
  • Combination immunizing preparation comprising recombinant plasmid and duplicate deficit type recombinant adenovirus
  • Combination immunizing preparation comprising recombinant plasmid and duplicate deficit type recombinant adenovirus

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1 Construction and Identification of Recombinant Plasmid pVR-lmp2 Containing LMP2 Sequence

[0047] In this example, the recombinant plasmid pVR-LMP2 containing the LMP2 sequence was constructed (see Figure 2 for the construction process), and the constructed recombinant plasmid was identified by recombinant plasmid sequencing and recombinant protein expression.

[0048] 1.1 Preparation of Escherichia coli DH5α competent cells

[0049] Pick a single colony of DH5α from a fresh plate cultured at 37°C for 16-20 hours, inoculate it in 5 ml of LB medium without antibiotics, and culture it overnight (12-16 hours) at 37°C with vigorous shaking. On the next day, draw 0.5ml from the above-mentioned culture and transfer it to 50ml LB medium at a ratio of 1:100 to continue culturing for about 3 hours until the OD600 value of the bacterial solution is 3. , In an ice-precooled 50ml centrifuge tube, ice bath for 30min. Centrifuge at 4000rpm for 10min at 4°C, discard the su...

Embodiment 2

[0084] Construction of Ad5 Replication Deficient Recombinant Adenovirus Containing LMP2 Sequence

[0085] In this example, a replication-defective recombinant adenovirus Ad5-LMP2 containing the LMP2 sequence was constructed.

[0086] 2.1 Construction of Ad5 replication-deficient recombinant adenoviral shuttle plasmid with LMP2 sequence

[0087] (1) Transformation of PSG5-LMP2 plasmid:

[0088] Pipette 1 μl PSG5-LMP2 plasmid with a sterile tip and add it to 200 μl DH5α competent cells, swirl gently to mix well, and place in ice bath for 30 minutes. Move the tube into a water bath at 42°C for 90 seconds, then quickly transfer the tube to an ice bath to cool the cells for 1-2 minutes. Add 800 μl of LB medium without antibiotics to each tube, move to a shaker at 37° C., and incubate for 45 minutes (rotational speed <150 rpm). Take 50 μl of transformed competent cells, use a sterile elbow glass rod to lightly coat the surface of the agar plate containing the correspo...

Embodiment 3

[0113] Identification, viral titer analysis and protein expression of a replication-deficient recombinant adenovirus Ad5-LMP2

[0114] 3.1 Transcription of LMP2 gene in 293 cells infected with recombinant adenovirus

[0115] TRIzol one-step method was used to extract the total RNA of normal 293 cells, Ad5-infected 293 cells and Ad5-LMP2-infected 293 cells for RT-PCR identification. RT-PCR reaction parameters are as follows: reverse transcription system is 20 μl. Use 1 μg of total cellular RNA as a template, Oligod(T)15 as a primer, perform reverse transcription reaction at 50°C for 30 minutes, inactivate reverse transcriptase at 94°C for 2 minutes, and perform PCR reaction with synthesized cDNA as a template, upstream primer P1: 5′GCTGCAGGAAACAACTCCCAATATCCA 3'; downstream primer P2: 5'AACTGGAGGGCAGCATCTAATGACC 3'. 30 cycles of 95°C for 30s, 55°C for 45s, and 72°C for 60s, 5 μl of RT-PCR reaction products were taken for agarose gel electrophoresis analysis. The results show...

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Abstract

The invention relates to a couple immunity agent which comprises recombine particle with EB virus hidden protein 2 gene and replication defect recombine adenovirus with said gene. The invention has significant cooperation function. Compared with single immunity, it can generate more specific CTL and specific antibodies, to effectively restrain and / or prevent EB virus cancer, as nasopharyngeal carcinoma.

Description

technical field [0001] The invention belongs to the fields of bioengineering and tumor treatment. The invention discloses a combined immunization preparation, which comprises a recombinant plasmid containing a gene encoding Epstein-Barr virus latent membrane protein 2 (LMP2) and a replication-deficient recombinant adenovirus containing the gene. Specifically, the present invention discloses a combined immunization preparation of recombinant plasmid pVR-LMP2 containing Epstein-Barr virus LMP2 coding gene and replication-deficient recombinant adenovirus Ad5-LMP2 containing the gene. The present invention further relates to the use of the combined immunization preparation and the recombinant plasmid in the preparation of drugs for preventing and treating Epstein-Barr virus-related tumors, and a treatment kit containing the combination immunization preparation. Background technique [0002] Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern Ch...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00A61P31/22A61P11/02C12N15/861C12M1/00
Inventor 曾毅周玲杨松梅王湛
Owner 中国疾病预防控制中心病毒病预防控制所
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