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Method for preparing capsid protein ú¿VPú®of enteric cytopathogenic human orphan virus 30

A capsid protein and virus technology, applied in the field of biomedicine, can solve the problems of low yield, high cost, and inability to be widely used in clinical practice.

Inactive Publication Date: 2008-07-23
昆山迪安医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, the preparation method of echovirus type 30 capsid protein is directly extracted from tissue cells, with low yield and high cost
Cannot be widely used clinically

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The preparation of embodiment 1 echovirus type 30 capsid protein

[0019] 1. Preparation of cDNA of pre-echovirus type 30 capsid protein (VP):

[0020] 2. Cloning preparation:

[0021] The expression clone of VP was prepared by classical methods of separation, cutting, splicing, transfection and screening.

[0022] The target gene expressing VP is amplified by PCR technique, and its product is purified. Then, the purified PCR product was digested with BamHI and HindIII, and inserted into pet41a which had been digested with the same double enzymes, to obtain a recombinant plasmid. Then the above plasmid was transformed into Escherichia coli DH5α, cultured and amplified, extracted and purified, and sequenced by an automatic sequencer. The recombinant plasmid with correct sequencing was transformed into BL21(DE3) bacteria, and cultured at 30° C. under the condition of 0.1 mM IPTG. After culturing, the bacterial fluid was lysed, and the protein bands were separated by S...

Embodiment 2

[0033] Application of embodiment 2 echovirus type 30 capsid protein 1

[0034] The echovirus type 30 capsid protein obtained by applying the method of the present invention is a new index with high sensitivity and high specificity, which can early distinguish bacterial and non-bacterial infections, and also contributes to the differential diagnosis of sepsis and MODS. The judgment of the prognosis of these diseases, the observation of curative effect and the monitoring of postoperative bacterial infection complications also have great clinical application value.

[0035] The echovirus type 30 capsid protein obtained by the invention is used to immunize animals to prepare antibodies, which can be made into diagnostic kits for the diagnosis, differential diagnosis, prognosis and curative effect observation of the above diseases.

Embodiment 3

[0036] Application 2 of embodiment 3 echovirus type 30 capsid protein

[0037] The finished echovirus type 30 capsid protein of the invention can be used in the preparation of clinical medicine, such as the candidate antigen of vaccine.

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PUM

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Abstract

This invention discloses a method for preparing ECHO virus type 30 VP. The method comprises: (1) performing trizol decomposition and extraction of ECHO virus type 30 to obtain VP mRNA, performing reverse transcription to obtain VP cDNA, amplifying by PCR, purifying and cutting with enzyme to obtain Pet41a+VP; (2) transferring to Escherichia coli DH5a, amplifying, extracting and purifying to obtain recombinant plasmid with right sequence; (3) transferring to BL21 (DE3) bacterium, and expressing recombinant protein at 30 deg.C and 0.1mM IPTG; (4) culturing the activated strains of recombinant protein, fermenting at a high density, and inducing in low-oxygen condition, decomposing the bacterium obtained, and dissolving the inclusion body and purifying; (5) inspecting by immunoassay or bioactivity detection, and collecting purified ECHO virus type 30 VP.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a preparation method of the specific antigen capsid protein (VP) of echovirus type 30. Background technique [0002] Echovirus (Echovirus) belongs to enteroviruses (Enteroviruses, EVs). Enteroviruses consist of 67 unique serotypes of viruses in the picornaviridae family. Picornaviruses are positive-strand RNA viruses that include polioviruses, coxsackieviruses groups A and B, echoviruses, and enteroviruses 68-71. Echovirus particles are very small and spherical. Virus particles are composed of simple capsids and single-sense RNA. They are naked without membranes and have a diameter of less than 30nm. The capsid of the virus contains four proteins, namely VP1, VP2, VP3, and VP4. Viral RNA is a single molecule, about 7.44kb in length, which is the template for viral translation and replication. There are 64 different serotypes of enteroviruses that can cause human infection, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/085
Inventor 周林福陈峰朱海红陈智
Owner 昆山迪安医学检验实验室有限公司
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