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Recombinant expression adenovirus fiber peptide, adenovirus subunit vaccine and preparation method of adenovirus cilia protein peptide

A technology of subunit vaccine and ciliary protein, which is applied in the direction of antiviral immunoglobulin, botany equipment and method, biochemical equipment and method, etc., can solve the problem of no report of cross-neutralization effect, and achieve convenient production and purification, Strong immunogenicity and high expression

Active Publication Date: 2018-01-19
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 adenovirus fiber peptide, adenovirus subunit vaccine and preparation method of adenovirus cilia protein peptide
  • Recombinant expression adenovirus fiber peptide, adenovirus subunit vaccine and preparation method of adenovirus cilia protein peptide
  • Recombinant expression adenovirus fiber peptide, adenovirus subunit vaccine and preparation method of adenovirus cilia protein peptide

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 the human adenovirus type 55 genome as a template, PCR was performed on A55Kn-RH / A55Kn-UB with primers to amplify the type 55 knob gene, and the PCR fragment was double-digested with BamHI and HindIII and connected to the above-mentioned prokaryotic expression vector PQE30 (Qiagen company), transformed Escherichia coli Top10 chemically competent, and picked a single colony for gene sequencing. Take colonies consistent with the theoretical sequence without mutations and extract plasmids.

[0042] A55Kn-RH: AGCTAATTAAGCttagtcgtcttctctgatgtagta (SEQ ID No: 13)

[0043] A55Kn-UB: ACCATCACGGAT...

Embodiment 2

[0053] The immunogenicity test of embodiment 2 recombinant protein

[0054] 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.

[0055] The result is as figure 2 and 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

[0056] Example 3 Mouse Anti-Adenovirus Ciliary Protein Head (knob) Serum Analysis of Cross-Neutralization of Different Types of Adenoviruses

[0057] 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.

[0058] Table 1 The cross-neutralization effect of mouse anti-adenovirus ciliary protein head serum on different types of adenovirus

[0059]

[0060] -: No neutralization detected

[0061] 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. Ad55 Knob can be used as a polyvalent adenovirus vaccine candidate against human adenovirus types 14, 3, and 55.

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Abstract

The invention discloses a recombinant expression adenovirus fiber peptide, an adenovirus subunit vaccine and a preparation method of the adenovirus fiber peptide. The recombinant expression adenovirusfiber peptide has an amino acid sequence shown in SEQ ID NO:2, and the vaccine comprises the above adenovirus fiber peptide. The adenovirus fiber peptide provided by the invention has a high expression quantity, when mice are immunized, a neutralizing antibody with a high titer can be induced, immunogenicity is strong, and the adenovirus fiber peptide can be used as an adenovirus subunit vaccinecandidate, is convenient for production and purification and low in costs; the adenovirus fiber peptide can induce a cross neutralization antibody, and can be used as a multivalent adenovirus vaccinecandidate; compatability of the adenovirus fiber peptide and other type knob can also be used as a multivalent adenovirus subunit vaccine candidate; and the adenovirus fiber peptide can be used as animmune antigen and a detection antigen for preparing a broad-spectrum neutralizing activity monoclonal antibody or a humanized 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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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/075C07K16/08C12N15/34C12N15/70A61K39/235A61K39/295A61P31/20
Inventor 田新贵周荣廖嘉仪周志超李潇
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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