Novel vaccine for preventing and treating Merkel cell carcinoma

A cell cancer, Merkel's technology, applied in the field of immunology and virology, can solve the problem of poor immune effect

Active Publication Date: 2021-08-20
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 poor

Method used

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  • Novel vaccine for preventing and treating Merkel cell carcinoma
  • Novel vaccine for preventing and treating Merkel cell carcinoma
  • Novel vaccine for preventing and treating Merkel cell carcinoma

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

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

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

[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 the plasmid pVAX-VP1 as a template, VP1 fragments containing endonuclease sites at the N-terminus and C-terminus were amplified respectively. The PCR reaction system is shown in Table 1 below.

[0097] Table 1 Amount of PCR reaction reagents

[0098]

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

[0100] Table 2 PCR reaction program

[0101]

[0102] (3) enzyme di...

experiment example 2

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

[0226] 1. Plasmid construction

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

[0228]Streak the pVAX1-VP1 / DH5α and pcDNA3.1-VP2 / DH5α glycerol bacteria on an LB plate (containing corresponding antibiotics such as Amp or Kan) and cultivate overnight in a 37°C incubator.

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

[0230] Plasmids were extracted from pVAX1-VP1 / DH5α and pcDNA3.1-VP2 / DH5α cultured overnight with a small plasmid extraction kit.

[0231] Using the plasmid pcDNA3.1-VP2 as the vector and pVAX1-VP1 ...

Embodiment 1

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

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

[0275] The specific steps of cloning are conventional methods in the art, and will not be repeated here. The cloning method includes PCR amplification, enzyme cutting, connection 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 conventional naming conventions in the art.

[0277] The method of identification is a conventional method in the art, 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 a conventional kit in the art, ...

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Abstract

The invention provides a novel vaccine for preventing and treating Merkel cell carcinoma. According to the novel vaccine for preventing and treating Merkel cell carcinoma, nucleotide sequences, such as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, having 71.9%-74.7% homology with SEQ ID No.6 are cloned and expressed, and the vaccine based on the Merkel cell carcinoma polyoma virus capsid protein VP1 is constructed. The problems of prevention and treatment of Merkel cell carcinoma are effectively solved, and great application value and application prospects are achieved.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(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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