Varicella-zoster virus gB-gE-gH-gL fusion protein, genetic engineering subunit vaccine and preparation methods

A technology of gb-ge-gh-gl, herpes zoster virus, applied in the field of biomedicine, which can solve the problems of increased possibility of virus reactivation infection, breakthrough infection, dangerous systemic infection, etc.

Active Publication Date: 2016-08-31
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recent studies have found that the vaccine has some shortcomings: 1. The protection rate of VZV live vaccine is less than 80%, and a small number of vaccinators may still be infected after close contact with chickenpox patients or VZV wild strains, said For "breakthrough inf...

Method used

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  • Varicella-zoster virus gB-gE-gH-gL fusion protein, genetic engineering subunit vaccine and preparation methods
  • Varicella-zoster virus gB-gE-gH-gL fusion protein, genetic engineering subunit vaccine and preparation methods
  • Varicella-zoster virus gB-gE-gH-gL fusion protein, genetic engineering subunit vaccine and preparation methods

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of gB-gE-gH-gL fusion protein

[0030]Prokaryotic expression of step 1 gB-gE-gH-gL fusion protein

[0031] The blister liquid collected from the skin blisters of chickenpox patients was inoculated onto MRC-5 cells, and the VZV virus was isolated and cultured. After the cells became lesions, the cells were collected, and the DNA was extracted by the phenol: chloroform: isoamyl alcohol method, and used as a PCR amplification method. Added templates;

[0032] According to the sequence of VZV Dumas (X04370.1) strain in Genebank, amino acid positions 136-285 of VZV gB protein, amino acid positions 37-161 of gE protein, amino acid positions 18-168 of gH protein, and amino acid positions of gL protein were respectively designed The nested PCR primers at positions 23-160 are connected with GGGGS between each two proteins, and NdeI and NocI restriction sites are reserved at both ends of the final gB-gE-gH-gL fusion gene;

[0033] The gB-gE-gH-gL fusion ge...

Embodiment 2

[0036] The immune effect detection of embodiment 2 subunit vaccine

[0037] Use the BCA method to detect the protein concentration of the purified gB-gE-gH-gL fusion protein, dilute it to 0.5mg / mL with PBS, filter it aseptically, and set aside; carry out the sterility test according to the current "Chinese Pharmacopoeia", and use the Limulus reagent method Endotoxin testing is carried out, and the endotoxin content is not higher than 100EU / mL before use;

[0038] Mix gB-gE-gH-gL fusion protein with an equal volume of aluminum hydroxide adjuvant or Freund's complete adjuvant 1:1, fully emulsify, and immunize 6-week-old BALB / c mice intraperitoneally, 25 μg / mouse Mice; 2 weeks after the initial immunization, booster immunization once, the dose was the same as the first time; at the same time, the same dose of recombinant VZV gE protein and PBS was inoculated as a control.

[0039] Three mice were sacrificed at the 3rd, 5th, and 7th weeks after inoculation, and the neutralizing a...

Embodiment 3

[0044] Embodiment 3 challenge protection test

[0045] A VZV strain that can adapt to guinea pig cells was cultivated in vitro, and then the peripheral blood lymphocytes of guinea pigs were infected with the virus strain in vitro, and then the guinea pig lymphocytes infected with VZV were reinfused into guinea pigs, and 28 days later, the intestinal ganglion of guinea pigs and establish a latent infection in the dorsal root ganglia. Utilize this model to carry out challenge protection test, divide 8 weeks old female Hartley guinea pigs 12 into two groups, every group 6, the first group is immune group, carries out secondary immunization with VZV genetic engineering subunit vaccine prepared by the present invention ( The second immunization was carried out 14 days after the first immunization, each time 100 μg per mouse, subcutaneous injection), the second group was the normal saline control group, and the same volume of sterile normal saline was injected. 28 days after the se...

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Abstract

The invention relates to the technical field of biomedicine, and particularly provides varicella-zoster virus gB-gE-gH-gL fusion protein, a genetic engineering subunit vaccine and preparation methods. The fusion protein comprises a VZV gB extracellular region, a gE extracellular region, a gH truncated fragment and a gL truncated fragment. The amino acid sequence of encoded protein of the fusion protein is SEQ ID NO:1, and one amino acid sequence is SEQ ID NO:2. A prokaryotic expression vector is utilized for constructing escherichia coli BL21 (DE3) host bacteria capable of expressing the VZV gB-gE-gH-gL fusion protein. The fusion protein is purified and mixed with a medicinal adjuvant to be prepared into the genetic engineering subunit vaccine. Compared with a currently-used VZV attenuated live vaccine, the vaccine can induce immune mice to generate higher specific humoral immunity and cell-mediated immunity and can prevent dormant infection that VZV is spread to dorsal root ganglions and intestinal ganglions through blood flow, the safety of the VZV vaccine is effectively improved, and therefore the genetic engineering subunit vaccine is an alternative vaccine with potential clinical application value.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the research of varicella-zoster virus gB-gE-gH-gL fusion protein, genetic engineering subunit vaccine and preparation method. Background technique [0002] Varicella-zoster virus (VZV) is a double-stranded DNA virus belonging to the α subfamily of herpesviruses. Chickenpox caused by VZV is extremely contagious, and the positive rate of antibodies in the serum of teenagers in my country has reached more than 85%. If chickenpox occurs in adults, severe visceral infection or even systemic infection often occurs. In the primary infection, the virus enters the neurons through the blood or the skin-nerve retrograde route to establish a latent infection, and the reactivation of the latent virus in the future can lead to herpes zoster. It is estimated that 1 / 3 of adults will have herpes zoster at least once, They have the potential for very painful long-term chronic pain. [00...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/295A61K39/25A61P31/22
CPCA61K39/12A61K2039/54C07K14/005C07K2319/00C12N2710/16722C12N2710/16734
Inventor 王明丽甘霖陈敬贤
Owner ANHUI MEDICAL UNIV
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