Prokaryotic expression of enterovirus 71 type VP1 (virus protein 1) and vaccine containing VP1

An enterovirus and prokaryotic expression technology, applied in the fields of genetic engineering and pharmacy, can solve the problems of poor immunogenicity of specific antibodies, pathogenicity, and the safety of genetically modified food needs to be evaluated, and achieve the effect of low cost and simple operation.

Inactive Publication Date: 2012-02-01
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from the current research stage, both the Salmonella vector vaccine and the live attenuated vaccine have the risk of causing disease; the edible vaccine expresses the EV71 virus VP1 protein in fruits, su

Method used

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  • Prokaryotic expression of enterovirus 71 type VP1 (virus protein 1) and vaccine containing VP1
  • Prokaryotic expression of enterovirus 71 type VP1 (virus protein 1) and vaccine containing VP1
  • Prokaryotic expression of enterovirus 71 type VP1 (virus protein 1) and vaccine containing VP1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of EV71 virus VP1 protein prokaryotic expression vector and expression and purification of VP1 protein

[0037] 1. Extraction of nucleic acid of EV71 virus HN-8 isolate and acquisition of VP1 protein gene sequence

[0038] Genomic RNA of EV71 virus was extracted by QIAamp Viral RNA Minikit Viral Nucleic Acid Extraction Kit from QIAGEN. The genomic RNA of EV71 virus was reverse-transcribed into cDNA using Transcriptor highfidelity cDNA systhesis sample Kit from Roche Company. The gene sequence of VP1 protein was amplified with a pair of primers: Nde1-VP1-F: 5'-CGCCATATGGGAGATAGGGTGGCAGATG-3' and Xho1-VP1-R: 5'-CCGCTCGAGAAGAGTGGTGATCGCTGT-3'. A Nde1 restriction site and protective bases were added to the 5' end of the VP1 fragment, and an Xho1 restriction site and corresponding protective bases were added to the 3' end of the amplified product. The PCR product was subjected to 1.2% agarose gel electrophoresis, the target band where the VP1 fragmen...

Embodiment 2

[0048] Example 2 Compatibility of VP1 protein and adjuvant to prepare vaccine

[0049] (1) Oral vaccine containing VP1 protein and its immunological effect evaluation

[0050] 1. VP1 protein and chitosan adjuvant are mixed and compatible

[0051] 1) Dissolve 0.25ml of glacial acetic acid in 100ml of double distilled water to obtain 0.25% acetic acid solution;

[0052] 2) Weigh 0.5 g of pharmaceutical grade acetylated chitosan (85% acetylated), dissolve it in 100 ml of 0.25% acetic acid solution, and obtain a 0.5 g / ml chitosan solution;

[0053] 3) Then add sodium polyphosphate with a final concentration of 0.05%, and mix at room temperature to obtain a colloidal solution containing chitosan microparticles, which is chitosan microparticles.

[0054] The VP1 protein is mixed with the prepared chitosan solution, the final concentration of chitosan in the vaccine is 0.2g-0.5g / ml, and the final volume is 1ml.

[0055] 2. Functional analysis of the vaccine

[0056] VP1 protein 1...

Embodiment 3

[0065] Example 3 Mucosal vaccine compatible with VP1 protein and different adjuvants and its immunological effect

[0066] (1) Immunization program

[0067] Select 4-5 weeks old Balb / C female mice (15±1g). Divide into 9 groups altogether, each group has 6; Set control group 3 groups (E-G), the mucosal vaccine immunization of different adjuvant compatibility is as shown in Table 1:

[0068] Table 1 Compatibility of VP1 protein with different adjuvants to immunize mice

[0069]

[0070] Group A: It is the whole virus control group, 100 μ g of inactivated EV71 virus is compatible with chitosan adjuvant to make a vaccine with a total volume of 1 ml;

[0071] Group B1: low-dose VP1 oral vaccine group, 100 μg of purified VP1 protein was mixed with chitosan adjuvant to make a vaccine with a total volume of 1 ml;

[0072] Group B2: VP1 oral vaccine high-dose group, 100 μg of purified VP1 protein is compatible with chitosan adjuvant to make a vaccine with a total volume of 1 ml; ...

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Abstract

The invention provides a prokaryotic expression method of an enterovirus 71 type VP1 (virus protein). The method comprises the steps of: cloning an enterovirus 71 type VP1 gene sequence into a prokaryotic expression carrier, transforming into coli bacillus to be expressed in an induction way, and purifying and renaturing the expressed VP1. The obtained VP1 and an immunologic adjuvant are matched with each other to prepare a vaccine. The invention has the advantages that: a genetic engineering recombinant VP1 is firstly expressed by a coli bacillus expression system, the operation is simple, the cost is low, large-scale production is easy to achieve and the like. The prokaryotic-expressed genetic engineering recombinant VP1 is matched with a chitosan adjuvant to immunize a mouse, and an immunological effect is evaluated, so that the VP1 is primarily proved to be not only capable of inducing the generation of a humoral immune protective reaction but also capable of inducing the generation of a cellular immunity reaction, therefore, the invention lays a foundation for further developing the genetic engineering recombinant VP1 and inactivated totivirus oral vaccine for human.

Description

technical field [0001] The invention relates to the fields of genetic engineering and pharmacy, in particular to the prokaryotic expression of enterovirus 71 type VP1 protein and a vaccine containing the protein. Background technique [0002] Enterovirus (Enterovirus) type 71 (EV71) prevalence in my country has been increasing year by year in recent years, and the main disease caused by EV71, hand-foot-mouth disease (HFMD), was included in my country's statutory regulations in 2008. Infectious diseases, in recent years, the incidence and death of HFMD have risen rapidly. Currently, symptomatic treatment is mainly adopted in clinical treatment, and there is no report on the application of vaccine in clinical treatment of HFMD. [0003] The EV71 virus vaccines currently in the research stage can be divided into injectable vaccines and oral vaccines according to the immunization route. Injectable vaccines include inactivated EV71 virus vaccines, genetically engineered recombin...

Claims

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Application Information

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IPC IPC(8): C12N15/70A61K39/125A61P31/14C12R1/19
Inventor 李德新张福顺梁米芳张硕张全福李川
Owner 中国疾病预防控制中心病毒病预防控制所
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