CN101906151A - Specific synthetic polypeptide for detecting novel H1N1 influenza viruses - Google Patents

Abstract

The invention discloses a specific synthetic polypeptide for quickly detecting novel H1N1 influenza viruses. The sequence of the polypeptide is CKYVKSTKLRLATGLRNVPS. The specific synthetic polypeptide has the advantages of simple antigen preparation, no biological safety problem, stronger specificity than natural microorganism or parasite protein antigen, low false negative rate and background reaction of detection antibodies and the like. In consideration of the diagnostic method of influenza viruses and the grim situation of prevention and control of influenza around the world currently, the polypeptide, undoubtedly, has relatively high market value and social benefit.

Description

A kind of synthetic polypeptide that is used for the specific detection novel H 1 N 1 influenza viruses

Technical field

The present invention relates to a kind of synthetic polypeptide that is used for the specific detection novel H 1 N 1 influenza viruses.

Background technology

Influenza virus is called for short influenza virus, is the Respirovirus that a class has hyperinfection, also is be put into the bio-terrorism weapon wherein a kind of.The method that detects novel H 1 N 1 influenza viruses at present mainly contains viral separation and Culture, reverse transcription poly integrated enzyme reaction (RT-PCR), serodiagnosis and Detection of antigen.But, required time of influenza virus isolation identification is long, cost is high, and need specify research institution to carry out at biocontainment laboratory or country, RT-PCR then depends on specific equipment, better laboratory condition and high-quality technician, be difficult to apply at border port, therefore, demand research urgently and set up a kind of novel H 1 N 1 influenza viruses diagnostic antigen technology fast and effectively.

Summary of the invention

Problem at prior art exists the object of the present invention is to provide a kind of synthetic polypeptide that is used for the specific detection novel H 1 N 1 influenza viruses.

For achieving the above object, specificity synthetic peptide sequence of the present invention is: CKYVKSTKLRLATGLRNVPS.

The screening method of the synthetic polypeptide of above-mentioned specificity is:

The aminoacid sequence of (1) retrieval from ncbi database, collection influenza virus HA hypotype, utilize DNA-STAR to carry out the comparison of sequence alignment and homology analysis, filter out in the HA molecule after the signal peptide to having 330-350 section that good resistance originality, wetting ability and processable analyze and 500-520 section between the protease cutting site as candidate segment;

(2) with the candidate's peptide section and the carrier protein couplet that filter out, obtain immune antibody,, obtain that finally novel H 1 N 1 influenza viruses is had specific synthetic polypeptide by the screening of specific antibody, the site of this polypeptide is 322-340, and its sequence is: CKYVKSTKLRLATGLRNVPS.

Further, described carrier proteins is the KLH hemocyanin.

Above-mentioned steps (2) is specially:

Detected immune serum with bag by the coupling polypeptide of immunizing rabbit,, carry out whole bloodletting, results serum when rabbit polyvalent antibody titre has reached 1: 100000;

Utilize totivirus (H3N2, H1N1, novel H1N1) to screen as the polyclonal serum antibody of envelope antigen to immunize rabbit, wherein,

Coating buffer: take by weighing Na ₂CO ₃0.15g, NaHCO ₃0.293g distilled water diluting obtains 0.05mol/L pH9.6 carbonic acid buffer to 100ml, 4 ℃, preserves;

Washings (diluent): take by weighing NaCl 8g, KH ₂PO ₄0.2g, Na ₂HPO ₄.12H ₂O 2.9g is measured Tween-20, and 0.5ml adds to 1000ml with distilled water, is 0.01mol/L pH7.4PBS-Tween-20 washings, 4 ℃, preserves;

Confining liquid: the pH7.4PBS of 5% skim-milk or BSA;

The sulphuric acid soln of stop buffer: 2mol/L;

Carry out ELISA experiment by known way then, and filter out according to experimental result novel H 1 N 1 influenza viruses is had specific synthetic polypeptide, that is:

1) envelope antigen: with coating buffer virus is done 1000 times of dilutions, the every hole of Sptting plate adds 100ul, and 4 ℃ of refrigerators are preserved 12h at least;

2) washing: liquid in the plate hole to the greatest extent, fill it up with washings, leave standstill 2min, three times repeatedly, at last Sptting plate is upside down on the thieving paper, make that washings flows to end in the hole;

3) add confining liquid 200ul, place 1h for 37 ℃;

4) washing is with 2);

5) add antibody: 1: the 500 times of dilution of confining liquid of rabbit immune serum, the every hole of Sptting plate adds 100ul, does the diluent contrast simultaneously, places 1h for 37 ℃;

6) washing is with 2);

7) add horseradish peroxidase goat anti-rabbit igg (1: 1000 times of dilution), the every hole 100ul of Sptting plate places 1h for 37 ℃;

8) washing is with 2);

9) add substrate: add TMB100ml, 15min is placed in the room temperature dark place;

10) add stop buffer: the every hole 50ul of Sptting plate;

11) observations: with enzyme-linked immunosorbent assay instrument record 490nm reading.

It is simple that the synthetic polypeptide of specificity of the present invention has antigen prepd, there is not the Biosafety problem, and than natural microbial or the antigenic high specificity of parasite protein, advantages such as the false negative rate of detection antibody and background reaction are all very low, the diagnostic method situation of comprehensive influenza virus, and the severe situation of current global prevention and control influenza, the present invention has bigger marketable value and social benefit undoubtedly.

Embodiment

The present invention is described in detail below in conjunction with example.

The screening method of the synthetic polypeptide of specificity of the present invention comprises the steps:

(1) information biology of peptide epitope is selected and synthetic, modification

The HA aminoacid sequence of search influenza virus from ncbi database is analyzed with known dna-STAR and antheprot5.0 and to be shown, between the 330-350 part antigenicity, wetting ability still be the processable analysis all be good candidate segment; Have a large amount of α spiral results in 400 downstreams, and mainly be positioned at the hydrophobicity fragment, and this section degree of variation is very low, sequence is stable, therefore is judged as the possible membrane portions of wearing, and abandon in this zone.But the short bigger zone of variation should zone (500-520) be arranged afterwards, and this incomplete antigen and wetting ability are all very strong.Simultaneously, the sequence of the epi-position linear peptides of each hypotype of influenza virus as shown in Table 1 and Table 2.

Table 1

Table 2

So in 330-350 and 500-520 zone, select synthetic ten alternative polypeptide of otherness fragment of different subtype, see Table 3.

Table 3

(2) preparation of antibody

Detected immune serum with bag by the coupling polypeptide of immunizing rabbit, when rabbit polyvalent antibody titre has reached 1: 100000, carry out whole bloodletting, results serum, the immune serum antibody titer of each polypeptide reach 1: 100000 o'clock P/N value and see Table 4.

Table 4

The immune serum numbering	Corresponding polypeptide title	The site	The gene type	1: 100000 o'clock PN value
					KLH-H5N1-1	1#	320-338	H5N1	82.1
KLH-H1N1-1	2#	321-339	H1N1	9.09
					KLH-H3N2-1	3#	238-256	H3N2	48.0
KLH-H2N2-1	4#	318-336	H2N2	6.02
					KLH-H5N1-2	ZS-5	498-520	H5N1	41.2
KLH-H1N1-2	ZS-6	495-517	H1N1	24.9
					KLH-H3N2-2	ZS-7	497-519	H3N2	34.6
KLH-H2N2-2	ZS-8	492-514	H2N2	27.9
					KLH-NH1N1-2	X1	496-518	New H1N1	18.6
KLH-NH1N1-1	X2	322-340	New H1N1	31.2

(3) screening of specific antibody in the synthetic polypeptide immune serum

The foundation of ELISA experiment

Utilize totivirus (H3N2, H1N1, novel H1N1) to screen as the polyclonal serum antibody of envelope antigen to immunize rabbit.

1. experiment material and reagent

H1N1, H3N2, new H1N1virus vaccine strain are from the Changchun institute of Biological Products.

Enzyme mark goat anti-rabbit igg antibody is available from Sigma company

96 hole enzyme plates available from glad through company of section

Bovine serum albumin (BSA) is available from Roch company

Tween20 is available from Sigma company.

Coating buffer: take by weighing Na ₂CO ₃0.15g, NaHCO ₃0.293g distilled water diluting is 0.05mol/L pH9.6 (coating buffer) carbonic acid buffer to 100ml, 4 ℃, preserves;

Washings (diluent): take by weighing NaCl 8g, KH ₂PO ₄0.2g, Na ₂HPO ₄.12H ₂O 2.9g is measured Tween-20, and 0.5ml adds to 1000ml with distilled water, is 0.01mol/L pH7.4PBS-Tween-20 washings (diluent), 4 ℃, preserves;

Confining liquid: the pH7.4PBS of 5% skim-milk or BSA;

The sulphuric acid soln of stop buffer: 2mol/L.

2.ELISA step

1) envelope antigen: with coating buffer virus is done 1000 times of dilutions, the every hole of Sptting plate adds 100ul, and 4 ℃ of refrigerators are preserved 12h at least;

2) washing: liquid in the plate hole to the greatest extent, fill it up with washings, leave standstill 2min, three times repeatedly, at last Sptting plate is upside down on the thieving paper, make that washings flows to end in the hole;

3) add confining liquid 200ul, place 1h for 37 ℃;

4) washing is with 2);

5) add antibody: 1: the 500 times of dilution of confining liquid of rabbit immune serum, the every hole 100ul of Sptting plate does the diluent contrast simultaneously, places 1h for 37 ℃;

6) washing is with 2);

7) add horseradish peroxidase goat anti-rabbit igg (1: 1000 times of dilution), every hole 100ul places 1h for 37 ℃;

8) washing is with 2);

9) add substrate: add TMB100ml, 15min is placed in the room temperature dark place;

10) add stop buffer: every hole 50ul;

11) observations: with enzyme-linked immunosorbent assay instrument record 490nm reading.

3.ELISA experimental result sees Table 5

Table 5

Interpretation as a result: if P/N＞2.1 o'clock then can be judged to be the positive.As can be seen from Table 5, the P/N value of immune serum KLH-NH1N1-1 and novel H1N1 reaction is 6.09, compares with other the P/N value of serum, and very big difference is arranged.Simultaneously, the P/N value of serum KLH-NH1N1-1 and viral H1N1 and H3N2 is judged to feminine gender all less than 2.1.Explanation thus, immune serum KLH-NH1N1-1 can specific and novel H1N1 virus reaction.Therefore, the serum by immunity after synthetic polypeptide X2 and the KLH coupling obtains can be used for the specific detection novel H 1 N 1 influenza viruses.

Claims (4)

1. the synthetic polypeptide of specificity that is used for the rapid detection novel H 1 N 1 influenza viruses is characterized in that the sequence of the synthetic polypeptide of described specificity is: CKYVKSTKLRLATGLRNVPS.

2. the screening method of the synthetic polypeptide of the described specificity of claim 1 is characterized in that, and is specific as follows:

The aminoacid sequence of (1) retrieval from ncbi database, collection influenza virus HA hypotype, utilize known dna-STAR to carry out the comparison of sequence alignment and homology analysis, filter out in the HA molecule after the signal peptide to having 330-350 section that good resistance originality, wetting ability and processable analyze and 500-520 section between the protease cutting site as candidate segment;

(2) with the candidate's peptide section and the carrier protein couplet that filter out, obtain immune antibody,, obtain that finally novel H 1 N 1 influenza viruses is had specific synthetic polypeptide by the screening of specific antibody, the site of this polypeptide is 322-340, and its sequence is: CKYVKSTKLRLATGLRNVPS.

3. screening method as claimed in claim 2 is characterized in that, described carrier proteins is the KLH hemocyanin.

4. screening method as claimed in claim 2 is characterized in that, described step (2) is specially:

Detected immune serum with bag by the coupling polypeptide of immunizing rabbit,, carry out whole bloodletting, results serum when rabbit polyvalent antibody titre has reached 1: 100000;

Utilize totivirus (H3N2, H1N1, novel H1N1) to screen as the polyclonal serum antibody of envelope antigen to immunize rabbit, wherein,

Coating buffer: take by weighing Na ₂CO ₃0.15g, NaHCO ₃0.293g distilled water diluting obtains 0.05mol/L pH9.6 carbonic acid buffer to 100ml, 4 ℃, preserves;

Washings (diluent): take by weighing NaCl 8g, KH ₂PO ₄0.2g, Na ₂HPO ₄.12H ₂O 2.9g is measured Tween-20, and 0.5ml adds to 1000ml with distilled water, is 0.01mol/L pH7.4PBS-Tween-20 washings, 4 ℃, preserves;

Confining liquid: the pH7.4PBS of 5% skim-milk or BSA;

The sulphuric acid soln of stop buffer: 2mol/L;

Carry out ELISA experiment by known way then, and filter out according to experimental result novel H 1 N 1 influenza viruses is had specific synthetic polypeptide, that is:

1) envelope antigen: with coating buffer virus is done 1000 times of dilutions, the every hole of Sptting plate adds 100ul, 4 ℃ of refrigerator overnight (more than the 12h);

2) washing: liquid in the plate hole to the greatest extent, fill it up with washings, leave standstill 2min, three times repeatedly, at last Sptting plate is upside down on the thieving paper, make that washings flows to end in the hole;

3) add confining liquid 200ul, place 1h for 37 ℃;

4) washing is with 2);

5) add antibody: 1: the 500 times of dilution of confining liquid of rabbit immune serum, the every hole of Sptting plate adds 100ul, does the diluent contrast simultaneously, places 1h for 37 ℃;

6) washing is with 2);

7) add horseradish peroxidase goat anti-rabbit igg (1: 1000 times of dilution), the every hole 100ul of Sptting plate places 1h for 37 ℃;

8) washing is with 2);

9) add substrate: add TMB100ml, 15min is placed in the room temperature dark place;

10) add stop buffer: the every hole 50ul of Sptting plate;

11) observations: with enzyme-linked immunosorbent assay instrument record 490nm reading.