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

A subunit vaccine, ciliated protein technology, applied in antiviral immunoglobulins, viral peptides, botanical equipment and methods, etc., can solve the problem of unreported cross-neutralization, achieve convenient production and purification, and high expression , strong immunogenic effect

Active Publication Date: 2017-12-12
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 cilia protein peptide, adenovirus subunit vaccine and preparation method of recombinant expression adenovirus cilia protein peptide
  • Recombinant expression adenovirus cilia protein peptide, adenovirus subunit vaccine and preparation method of recombinant expression adenovirus cilia protein peptide
  • Recombinant expression adenovirus cilia protein peptide, adenovirus subunit vaccine and preparation method of recombinant expression 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 with the primer pair A55Kn-RH / A55Kn-UB to amplify the type 55 knob gene, and the PCR fragment was double-digested with BamHI and HindIII and connected to the above-mentioned PQE30 to transform Escherichia coli Top10 chemically competent, pick 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:ACCATCACGGATCCaacaacatttgcattgatgac (SEQ ID No: 14)

[0044]...

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 all 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]

[0061] -: No neutralization detected

[0062] 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. Ad3 Knob antiserum can neutralize human adenovirus types 3, 14, and 55, so Ad3 Knob can be used as a multivalent adenovirus vaccine candidate for preventing human adenovirus types 3, 14, and 55.

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Abstract

The invention discloses recombinant expression adenovirus cilia protein peptide, an adenovirus subunit vaccine and a preparation method of the recombinant expression adenovirus cilia protein peptide. The recombinant expression adenovirus cilia protein peptide has an amino acid sequence shown by SEQ ID No:2, and the vaccine contains the adenovirus cilia protein peptide. The adenovirus cilia protein peptide disclosed by the invention has advantages of high expression quantity, strong immunogenicity, capability of serving as a candidate of the adenovirus subunit vaccine, convenience in production and purification and low cost; by means of the adenovirus cilia protein peptide, an immune mouse can induce a high-titer neutralizing antibody; the adenovirus cilia protein peptide can induce a cross neutralizing function antibody and can serve as a candidate of a polyvalence adenovirus vaccine; when being matched with other types of knob, the adenovirus cilia protein peptide also can serve as a candidate of a polyvalence adenovirus subunit vaccine; the adenovirus cilia protein peptide can serve as immunizing antigen and detecting antigen for preparing a broad-spectrum neutralizing active monoclonal antibody or 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): C07K14/075C12N15/34C12N15/70A61K39/235A61P31/20C07K16/08
CPCA61K39/12A61K2039/55505A61K2039/70C07K14/005C07K16/081C12N15/70C12N2710/10022C12N2710/10034C12N2800/22
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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