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EV71 subunit vaccine of mixed adjuvant and preparation method thereof

A subunit vaccine and adjuvant technology, applied in the field of biomedicine, can solve the problems of low titer of neutralizing protective antibodies, and achieve the effects of easy large-scale production, low cost and simple preparation process

Inactive Publication Date: 2016-05-04
BEIJING KYNING BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to overcome the defect that the neutralizing protective antibody titer that existing EV71 subunit vaccine produces is low, provide a kind of EV71 subunit vaccine preparation method with mixed adjuvant, use the unlabeled VP1 protein of EV71 as Antigen, protein combined with aluminum adjuvant first, washed and then mixed with MPLA adjuvant to prepare EV71 subunit vaccine

Method used

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  • EV71 subunit vaccine of mixed adjuvant and preparation method thereof
  • EV71 subunit vaccine of mixed adjuvant and preparation method thereof
  • EV71 subunit vaccine of mixed adjuvant and preparation method thereof

Examples

Experimental program
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Embodiment 1

[0045] 1. Synthesis of VP1 gene and construction of recombinant vector pET22b-VP1

[0046] The EV71 virus strain derived from the C4 subtype was obtained from the Pasteur Institute in Shanghai, the viral RNA was extracted with a viral RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), and the viral cDNA was obtained by reverse transcription with a reverse transcription kit. Design primers to amplify the full sequence of the virus in segments, digest and link the segmented sequences into a complete sequence, and send it to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing. The sequence of VP1 is finally determined as shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2 , and this full-length cDNA sequence has also repackaged a new infectious virus through in vitro transcription, so it is confirmed that the cDNA sequence is complete and the function is not lost.

[0047] Based on the VP1 sequence obtained by the above sequencing as a standa...

Embodiment 2

[0071] 1. Mix different immune adjuvants and VP1 antigen protein

[0072] The protein sample collected through the gel filtration chromatography column was first combined with aluminum hydroxide adjuvant, and the solution of the protein sample was (20mmol / LTris-HClpH8.0, 8mol / L urea, 70mmol / Lβ-mercaptoethanol), the protein The concentration is about 0.5mg / ml, the volume is about 200μl, the amount of aluminum hydroxide adjuvant is 200μg, mix and stand at room temperature for 30min, mix with a pipette tip in the middle, centrifuge at 3000rpm, remove the supernatant, and buffer with 10mmol / LpH7.0HEPES The antigenic protein coated with aluminum hydroxide adjuvant was obtained, which was the immunogen ①; then the coated protein was mixed with 20 μg MPLA (Invivogen) adjuvant, and supplemented with 10 mmol / L pH7.0 HEPES buffer to 200 μl , as the immunogen for mice ②; the protein sample purified by gel filtration chromatography was dialyzed and freeze-dried to obtain 100 μg protein sa...

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Abstract

The invention discloses an EV71 subunit vaccine of a mixed adjuvant and a preparation method thereof. According to the vaccine, C4 sub-type EV71 virus VP1 protein is used as an antigen, and aluminum hydroxide and monophosphoryl lipid A are used as a mixed adjuvant. VP1 protein which is expressed and purified by escherichia coli and does not have any label is used as an antigen; the prepared protein is combined with an aluminum adjuvant under a denaturing condition, is subjected to detergence determination, and is mixed with an MPLA adjuvant to immune mice; and the prepared polyvalent antibody can be used for specifically neutralizing the EV71 virus to generate a neutral protective antibody having a titer nearly 1:128. Compared with an EV71 inactivated vaccine in current clinical tests, the human enterovirus 71 type subunit vaccine has the characteristics of low cost, simple operation, high safety, easy large-scale production and the like in the production process. The vaccine can generate relatively good immunogenicity in a human body, has a relatively high titer of the neutralizing antibody, and is an alternative vaccine with potential clinical application value.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an EV71 subunit vaccine mixed with an adjuvant and a preparation method thereof. technical background [0002] Hand, foot and mouth disease is a common childhood disease characterized by fever, rashes, ulcers, and herpetic pharyngitis on the affected hands, feet, and mouth. A small number of patients can also cause fatal complications such as myocarditis, pulmonary edema, and encephalitis. , mostly occurs in infants under the age of 5. The pathogens causing HFMD are mainly Enterovirus 71 and Coxsackievirus A16. [0003] Studies have shown that EV71 is mainly related to severe hand, foot and mouth disease. It has neurotropic toxicity and further causes aseptic encephalitis, meningoencephalitis and myocarditis and other complications. In severe cases, it may be disabled or dead. Since EV71 was first isolated from fecal samples of patients with central nervous system diseases in Califor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/125A61P31/14C12N15/70C07K14/085C07K1/18C07K1/16
CPCA61K39/39A61K39/12A61K2039/545A61K2039/55505A61K2039/55572C07K14/005C12N2770/32034C12N2770/32322
Inventor 吴洪流胡广张明陈国郭蕾
Owner BEIJING KYNING BIOSCI
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