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A novel vaccine for the prevention and treatment of Merkel cell carcinoma

A cell carcinoma, Merkel's technology, applied in the fields of immunology and virology, can solve problems such as poor immune effect

Active Publication Date: 2022-06-28
ADVACCINE SUZHOU BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the vaccine only uses nucleic acid as the active ingredient of the vaccine, and the immune effect is relatively poor.

Method used

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  • A novel vaccine for the prevention and treatment of Merkel cell carcinoma
  • A novel vaccine for the prevention and treatment of Merkel cell carcinoma
  • A novel vaccine for the prevention and treatment of Merkel cell carcinoma

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0088] Basic experimental example 1 Construction, screening and verification of CMS5-VP1 tumor cell line

[0089] The CMS5-VP1 tumor cell line will be mainly used for the verification of vaccine efficacy.

[0090] 1. Construction of pcDH-VP1 plasmid

[0091] (1) First, design primers according to the pcDH-GFP plasmid:

[0092] MCV-F(5'-CTAGAGCTAGCGTTTAAACTTAAGC-3');

[0093] MCV-R (5'-GCGGCCGCTCTAGACTCGAGTCACAGCTCCTG-3').

[0094] The 5' end of MCV-F contains a Nhe I restriction site, and the 3' end of MCV-R contains a Not I restriction site.

[0095] (2) PCR amplification

[0096] Using plasmid pVAX-VP1 as a template, a VP1 fragment containing an endonuclease site at the N-terminus and C-terminus was obtained by amplification. The PCR reaction system is shown in Table 1 below.

[0097] Table 1 PCR reaction reagent dosage table

[0098]

[0099] The PCR reaction program is shown in Table 2 below.

[0100] Table 2 PCR reaction program

[0101]

[0102] (3) Enzyme...

experiment example 2

[0225] Basic experimental example 2MCV-VP1_VP2 pseudovirus construction, expression verification

[0226] 1. Plasmid construction

[0227] The plasmid pcDNA3.1-VP2 was synthesized by Nanjing GenScript, inserted into the multi-cloning site of vector pcDNA3.1 by endonuclease Kpn I / Xho I, and the correctness of the insertion site was confirmed by sequencing.

[0228] The pVAX1-VP1 / DH5α and pcDNA3.1-VP2 / DH5α glycerol bacteria were streaked on LB plates (containing corresponding antibiotics such as Amp or Kan), and cultured in a 37°C incubator overnight.

[0229] The pVAX1-VP1 / DH5α and pcDNA3.1-VP2 / DH5α monoclonal colonies were respectively picked and placed in 5 mL LB liquid medium (containing corresponding antibiotics such as Amp or Kan), and incubated overnight at 37°C with shaking on a shaker.

[0230] The plasmids of pVAX1-VP1 / DH5α and pcDNA3.1-VP2 / DH5α cultured overnight were extracted with plasmid mini-suction kit.

[0231] Using plasmid pcDNA3.1-VP2 as the carrier and pVA...

Embodiment 1

[0273] Example 1 A DNA vaccine against Merkel cell carcinoma

[0274] The nucleotide sequence shown in SEQ ID NO. 1 was cloned into the pVAX1 vector, and the enzyme cleavage sites used for the cloning were XbaI and HindIII.

[0275] The specific steps of cloning are conventional methods in the art, which will not be repeated here. Cloning methods include PCR amplification, restriction digestion, ligation and related steps.

[0276] The recombinant vector obtained by successfully cloning the nucleotide sequence shown in SEQ ID NO. 1 into the pVAX1 vector was named pVAX1-MCV-VP1 according to the conventional naming convention in the art.

[0277] The identification method is a conventional method in the field, and will not be repeated here. For example, double-enzyme digestion identification or sequencing identification. After double digestion with XbaI and HindIII, a DNA band of about 1.3kb can be observed.

[0278]pVAX1-MCV-VP1 was amplified using conventional kits in the ...

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Abstract

The invention provides a novel vaccine for preventing and treating Merkel cell carcinoma. The present invention is carried out by using a nucleotide sequence having 71.9-74.7% homology with SEQ ID No.6, such as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. Cloned and expressed, a vaccine based on Merkel cell carcinoma polyomavirus capsid protein VP1 was constructed. The invention effectively solves the problems of prevention and treatment of Merkel cell carcinoma, and has great application value and application prospect.

Description

technical field [0001] The invention belongs to the field of immunology and virology, and particularly relates to a novel vaccine for preventing and treating Merkel cell carcinoma. Background technique [0002] Merkel cell carcinoma polyomavirus (MCV) is currently the only known polyomavirus directly associated with human cancer. Merkel cell carcinoma polyoma virus is a double-stranded non-enveloped DNA virus with an icosahedral spherical structure of about 45 nm and a genome size of 5386 bp, containing early coding regions, late coding regions and non-coding regulatory regions ( NCRR). The early genes of Merkel cell carcinoma polyomavirus mainly encode LT, ST, 57kT and ALTO (alternating frame of large T open reading frame), while the late genes mainly encode the capsid proteins VP1 and VP2. VP1 has the function of initiating viral infection and often exists in the form of pentamers, and 72 pentamers can self-assemble into virus-like particles in eukaryotic systems. [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/37C07K14/025A61K39/12A61K39/39A61K48/00A61P35/00
CPCC07K14/005A61K39/12A61K39/39A61K48/005A61P35/00C12N2710/22022C12N2710/22034A61K2039/53A61K2039/876A61K2039/575A61K2039/55505A61K2039/55561A61K2039/55511Y02A50/30
Inventor 何悦赵干张璐楠程鑫俞庆龄
Owner ADVACCINE SUZHOU BIOPHARMACEUTICALS CO LTD
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