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Recombinant expression type adenovirus dynein peptide, adenovirus subunit vaccine and preparation method thereof

A subunit vaccine, ciliated protein technology, applied in antiviral immunoglobulins, viral peptides, botanical equipment and methods, etc., can solve the problem of no report on cross-neutralization, and achieve convenient production and purification, low cost, Strong immunogenic effect

Active Publication Date: 2017-11-21
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cross-neutralization between different types of Knob of group B adenovirus has not been reported

Method used

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  • Recombinant expression type adenovirus dynein peptide, adenovirus subunit vaccine and preparation method thereof
  • Recombinant expression type adenovirus dynein peptide, adenovirus subunit vaccine and preparation method thereof
  • Recombinant expression type adenovirus dynein peptide, adenovirus subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Expression of recombinant human adenovirus knob polypeptide

[0040] 1. Escherichia coli preferred codons were optimized for human type 3, type 7, type 14, type 4, and type 5 adenovirus knob genes to adapt to expression in E. coli, and the genes were synthesized separately and cloned into the prokaryotic expression vector PQE30 (Qiagen Company) middle;

[0041] 2. Using human type 11 and type 55 adenovirus genomes as templates, PCR was performed with primer pairs A11Kn-RH / A11Kn-UB and A55Kn-RH / A55Kn-UB to amplify type 11 and type 55 knob genes, BamHI and The PCR fragment was double-digested with HindIII and connected to the above-mentioned prokaryotic expression vector PQE30 (Qiagen Company), transformed into Escherichia coli Top10 chemically competent, and a single colony was picked for gene sequencing. Take colonies consistent with the theoretical sequence without mutations and extract plasmids;

[0042] A11Kn-RH: aattAAGCTTTAgtcgtcttctctgatgtagta (SEQ ...

Embodiment 2

[0056] The immunogenicity test of embodiment 2 recombinant protein

[0057] In order to analyze the immunogenicity of the recombinant protein of Example 1, the recombinant protein was added to the emulsified aluminum phosphate adjuvant (ADJU-PHOS, Brenntag Biosector) to immunize mice for four times, and each mouse was immunized with 40ug protein each time. Serum was collected 7 days after the last immunization for ELISA detection and cell microneutralization experiments.

[0058] The result is as figure 2 with image 3 As shown, the results show that various types of recombinant knob immunized mice can induce high-titer antibody responses, and the neutralizing antibody titers to respective types of adenoviruses are about 500-4000 ( figure 2 ), the titer of ELISA detection with recombinant protein antigen can reach 10 6 -10 7 ( image 3 ).

Embodiment 3

[0059] Example 3 Mouse Anti-Adenovirus Ciliary Protein Head (knob) Serum Analysis of Cross-Neutralization of Different Types of Adenoviruses

[0060] The anti-ciliated protein head serum of Example 2 after four times of immunization was collected for cross-neutralization detection, and the results are shown in Table 1.

[0061] Table 1 The cross-neutralization effect of mouse anti-adenovirus ciliary protein head serum on different types of adenovirus

[0062]

[0063] -: No neutralization detected

[0064] The results in Table 1 show that some types have cross-neutralizing effects on multiple types of adenoviruses in the same group, and this cross-neutralizing effect is very strong. Ad11 type Knob antiserum can neutralize human type 11, type 7, type 14, type 55 adenovirus. Therefore Ad11 type Knob can be used as a polyvalent adenovirus vaccine candidate against human type 11, 7, 14, and 55 adenoviruses.

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Abstract

The invention discloses recombinant expression type adenovirus dynein peptide, an adenovirus subunit vaccine and a preparation method thereof. The recombinant expression type adenovirus dynein peptide has an amino acid sequence shown as SEQ ID No: 2; the vaccine contains the adenovirus dynein peptide. The adenovirus dynein peptide, disclosed by the invention, has a high expression quantity, can be used for inducing a high-titer neutralizing antibody when being used for immunizing a mouse and has strong immunogenicity; the adenovirus dynein peptide can be used as a candidate of the adenovirus subunit vaccine, is convenient to produce and purity and is low in cost; the adenovirus dynein peptide can be used for inducing a cross neutralization effect antibody and can be used as a candidate of a multivalent adenovirus vaccine; the adenovirus dynein peptide can be matched with other types of knob to be used as a candidate of a multivalent adenovirus subunit vaccine; the adenovirus dynein peptide can also be used as an immune antigen and a detection antigen which are used for preparing broad-spectrum neutralization activity monoclonal antibody or a human-derived monoclonal antibody.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. More specifically, the invention relates to a recombinantly expressed adenovirus ciliary protein peptide, an adenovirus subunit vaccine, a multivalent adenovirus subunit vaccine and a preparation method thereof. Background technique [0002] Human Adenovirus (HAdV) can infect a variety of mucosal tissues, such as the gastrointestinal tract, respiratory tract, genitourinary tract, and cornea and conjunctiva, leading to acute respiratory infection (ARI), acute gastroenteritis, nephritis, and keratoconjunctivitis , cystitis and other self-limiting mucosal infections and even severe fatal infections. Adenoviruses are widespread and spread rapidly. In recent years, there have been outbreaks and epidemics in many countries and regions, which seriously threaten public health security. People of all ages can be infected by adenovirus, and most of the susceptible people are infants and immuno...

Claims

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Application Information

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IPC IPC(8): C07K14/075C07K16/08C12N15/34C12N15/70A61K39/235A61K39/295A61P31/20
CPCA61K39/12A61K2039/70C07K14/005C07K16/081C12N15/70C12N2710/10022C12N2710/10034
Inventor 田新贵周荣廖嘉仪李潇周志超
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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