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Human enterovirus 71 type specific polypeptide and application thereof

A human enterovirus, specific technology, applied in the field of medical immunity, can solve problems such as false positives

Inactive Publication Date: 2010-11-24
BEIJING KYNING BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, many countries in the world, including my country, implement universal vaccination against poliovirus, so the detection of human enterovirus 71 by the traditional whole-virus detection antibody method may produce false positives

Method used

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  • Human enterovirus 71 type specific polypeptide and application thereof
  • Human enterovirus 71 type specific polypeptide and application thereof
  • Human enterovirus 71 type specific polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Synthesis of a polypeptide

[0043] The method used to synthesize the polypeptides of SEQ ID NO. 1 to SEQ ID NO. 6 is a conventional chemical synthesis method.

[0044] b peptide screening

[0045] According to the amount of 10μg of the above polypeptide per well, dissolved in 100μl of coating solution (1.59g Na 2 CO 3 , 2.93g NaHCO 3 ) And coat the ELISA plate for 12 hours, discard the coating solution, wash with deionized water twice, pat dry on absorbent paper each time; add 250μl blocking solution (PBS, 0.05% Tween-20, 5%, skimmed milk powder) placed at room temperature for 2 hours; discard the blocking solution, add serum diluted 1:50 with 100μl blocking solution, the serum used is the mouse antiserum immunized with EV71 VP1 protein, Immune mouse antiserum with PV VP1 protein and unimmunized control serum; leave at room temperature for 1 hour; discard the liquid; wash 5 times with deionized water; add 100μl of blocking solution 1:5000 diluted biotin-labeled anti-mouse Ig...

Embodiment 2

[0048] Example 2 Genetic engineering expression of polypeptide.

[0049] SEQ ID NO. 8 is the nucleotide sequence encoding the polypeptide 1 of the present invention. The target gene is synthesized with this sequence, and the restriction sites of BamHI and HindIII are introduced respectively at both ends. The gene was inserted into the BamHI and HindIII sites of the vector pQE-80L (Qiajie Biotechnology (Shanghai) Co., Ltd.). The vector pQE-80L contains the gene sequence encoding the polypeptide of SEQ ID NO.7. The recombinant vector capable of accurately expressing the fusion protein of SEQ ID NO1 and SEQ ID NO7 is transformed into E. coli BL21 (DE3), and the obtained genetically engineered strain can be used to express the polypeptide of the present invention.

[0050] The expression conditions are as follows: single clones are selected and inoculated in 10 mL of LB medium, and cultured overnight at 37°C in a shaker. The bacterial solution was inoculated into an auto-induction me...

Embodiment 3

[0053] Polypeptide 1 was dissolved in 100μl coating solution (1.59g Na 2 CO 3 , 2.93gNaHCO 3 ) Coated ELISA plate for 12 hours, discard the coating solution, wash 2 times with deionized water, pat dry on absorbent paper each time; add 250μl blocking solution (PBS, 0.05) to each well %Tween-20, 5%, skimmed milk powder) placed at room temperature for 2 hours; discard the blocking solution, and add serum diluted with 100μl blocking solution 1:50. The serum used is the serum of the patient with EV71 infection and the normal serum. Healthy human serum, pregnant woman serum and control serum (a known healthy human serum); placed at room temperature for 1 hour; discarded the liquid; washed 5 times with deionized water; added 100μl of blocking solution diluted 1:5000 biotin label Anti-human IgM antibody secondary antibody, put it at room temperature for half an hour; discard the liquid, add 100μl of blocking solution 1:5000 diluted AP-labeled streptavidin, place at room temperature for ...

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PUM

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Abstract

The invention belongs to the field of medical immunology, and discloses human enterovirus 71 type specific polypeptide and application thereof. The amino acid sequence of the human enterovirus 71 type specific polypeptide, which is capable of triggering an immunological response to EV71, is derived from the polypeptide 1 derived and has an amino acid sequence SEQ ID NO.1 of an amino acid sequence which is formed by substituting, losing or adding 1 to 10 amino acid residues in amino acid sequence SEQ ID NO.1, or a polypeptide fragment which has the function of triggering the immunological response to EV71 and of which the length of the amino acid sequence is at least 15 continuous amino acid residues in the SEQ ID NO.1. The polypeptide provided by the invention can be specifically reacted with the blood serum of human enterovirus 71 type patients, and simultaneously not cross reacted with poliomyelitis viruses. Therefore, the human enterovirus 71 type specific polypeptide can be applied to clinical test and diagnosis of human enterovirus 71 type infection.

Description

Technical field [0001] The invention belongs to the field of medical immunity, and relates to a human enterovirus type 71 specific polypeptide and its application. technical background [0002] The main pathogens of hand, foot and mouth disease are Enterovirus 71 (EV71), Coxsackievirus A16 (Coxsackieviruses A16, CAV16), etc. However, EV71 is the main cause of neurological symptoms and death. EV71 was first isolated in 1969 from a stool specimen of an infant with central nervous system disease in California. Since it was first reported in 1974, outbreaks of varying degrees were caused in Bulgaria, Hungary, Malaysia and Taiwan in 1975, 1978, 1997 and 1998, involving dozens of deaths. It is worth mentioning that although developed countries including Japan and the United States occasionally have sporadic cases, there have been no reports of large-scale epidemics and outbreaks in decades. In March 2008, an outbreak of hand, foot and mouth disease began in Fuyang, Anhui Province, wh...

Claims

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Application Information

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IPC IPC(8): C07K14/085C07K16/10C12N15/41A61K39/42A61K39/125A61P31/14G01N33/569
CPCY02A50/30
Inventor 胡广金明飞冷启彬吴洪流
Owner BEIJING KYNING BIOSCI
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