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Construction method of multivalent epitope and subunit vaccine

A subunit vaccine and subunit technology, applied in the field of vaccines, to achieve the effect of a large number of immunogens and a rich variety

Pending Publication Date: 2020-09-01
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Second, mainly viral infectious diseases, but also face the dilemma that there is no specific medicine (including vaccines) to treat

Method used

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  • Construction method of multivalent epitope and subunit vaccine
  • Construction method of multivalent epitope and subunit vaccine
  • Construction method of multivalent epitope and subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of EP1-LTB26-EP2 fusion protein (bivalent vaccine):

[0034] 1. Recombinant expression

[0035] 1.1 Cloning and expression of pET32-RBD EP1-LTB26-RBD EP2 gene

[0036] a. Synthetic recombinant target gene:

[0037]Insert LTB26 gene (SEQ ID NO.1) between SacI (GAGCTC) and SalI (GTCGAC) restriction sites of the DNA sequence (SEQ ID NO.1) of the receptor binding domain (RBD) of the novel coronavirus (SARS CoV-2) ID NO.2), which is equivalent to connecting the epitopes EP1 (SEQ ID NO.3) and EP2 (SEQ ID NO.4) of the novel coronavirus at both ends of LTB26 to form an EP1-LTB26-EP2 fusion gene. Then, the EP1-LTB26-EP2 fusion gene was constructed on an ordinary Escherichia coli expression vector (such as pET32a).

[0038] The sequence involved is as follows:

[0039] DNA sequence encoding RBD (SEQ ID NO.1):

[0040]

[0041]

[0042] In SEQ ID NO.1, the underlined part indicates the enzyme cutting sites SacI (GAGCTC) and SalI (GTCGAC).

[0043]...

experiment example 2

[0065] Experimental example 2 Safety detection of EP1-LTB26-EP2 bivalent vaccine polypeptide of the present invention

[0066] 1. Experimental method

[0067] Taking male rats as an animal model, the EP1-LTB26-EP2 prepared in Example 1 of the present invention was injected intraperitoneally into the rats to detect its safety:

[0068] a) Six healthy male rats with a body weight of 3-4 weeks were obtained from the Experimental Animal Center of Chongqing Medical University.

[0069] b) Dissolving the EP1-LTB26-EP2 with immune adjuvant activity prepared in Example 1 with PBS buffer.

[0070] c) Add 0.50ml of EP1-LTB26-EP2 (without Exogenous endotoxin detection and treatment) were intraperitoneally injected into the rats described in a) above, and the differences in signs between the experimental rabbit and the control group were observed within 3 days (such as activity ability, feeding ability, whether there was tremor, vertical hair, loose stool, etc.), and the survival situa...

Embodiment 3

[0076] Embodiment 3 Effectiveness detection of EP1-LTB26-EP2 bivalent vaccine of the present invention

[0077] 1. Experimental method

[0078] The EP1-LTB26-EP2 fusion protein with immune adjuvant activity prepared in Example 1 was dissolved with PBS buffer. Follow the steps below to check the effectiveness:

[0079] a) 18 rats (male) were purchased from the Animal Experiment Center of Chongqing Medical University and randomly divided into 4 groups with 6 rats in each group.

[0080] b) The EP1-LTB26-EP2 fusion protein prepared in Example 1 (6.0 μg / rat) was immunized by nasal drops to 4% chloral hydrate anesthetized rats (1.0 ml / 100 g); PBS nasal drops were used as a control.

[0081] c) On the 7th day after the initial immunization, carry out the second booster immunization with the same dose as in b) above, and perform the third booster immunization on the 14th day. Before each booster immunization, blood samples should be collected and stored at -80°C for future use. On...

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Abstract

The invention discloses a construction method of a multivalent epitope and subunit vaccine, and belongs to the field of vaccines. An antigen protein is connected to two ends of LTB26 through fusion expression to obtain a fusion protein, and a pentamer of the fusion protein is obtained by means of the characteristic that LTB26 can be self-assembled to form a pentamer. The active ingredient of the vaccine is the pentamer of the fusion protein. The vaccine is rich in immunogen quantity and variety, and the activity of a LTB26 immunologic adjuvant and the immunogen activity of an antigen peptide are combined together, so that the vaccine can stimulate an organism to generate a large number of specific antibodies, effective immune response is stimulated, and a procedure of adding an immunologicadjuvant is omitted. The protein fused to two ends of LTB26 can also be other proteins other than an antigen protein, and can be applied to the fields other than vaccine preparation, such as large-scale preparation of antibodies, research and development of medical detection kits, and the like.

Description

technical field [0001] The present invention belongs to the field of vaccines. Background technique [0002] Immunization (immunization) is to inoculate the vaccine in the body of healthy people so that the vaccinators can produce antibodies against specific pathogens without getting sick, obtain specific immunity to specific diseases, and achieve the purpose of preventing and treating certain infectious diseases. According to different vaccination sites, it can be divided into traumatic vaccination (such as injection, scratching, etc.) and non-invasive vaccination (such as nasal drop, spray, etc.). [0003] At present, the research and development of vaccines mainly includes five technical routes: inactivated vaccines, live attenuated vaccines, recombinant genetic engineering vaccines, virus vector vaccines and nucleic acid vaccines [Jia Bingbing, Li Weiguo. History and Prospect of Vaccine Technology Development [J]. Biology Bulletin, 2016, 51(06): 1-3.]. Due to the long ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C07K19/00A61K39/215A61P31/14A61K39/39A61P37/04
CPCC07K14/245C07K14/005C12N15/70A61K39/12A61P31/14A61K39/39A61P37/04C12N2770/20022C12N2770/20034A61K2039/55516A61K2039/70
Inventor 马永平
Owner CHONGQING MEDICAL UNIVERSITY
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