Foot-and-mouth disease virus type a elisa antibody detection kit and preparation method and application thereof

Abstract

The application discloses a foot-and-mouth disease virus type A ELISA antibody detection kit and a preparation method and application thereof. The application determines a sequence of a foot-and-mouth disease virus type A GH loop epitope polypeptide and a non-specific combination of a foot-and-mouth disease virus type O, effectively solves the non-specific combination problem through sequence reduction, obviously reduces the detection sensitivity of the type A antibody after the reduction, further improves the detection sensitivity through a form of a multi-antigen peptide to display the polypeptide sequence. Finally, the polypeptide epitope combination, the antigen coated plate containing the polypeptide epitope combination and the ELISA antibody detection kit are determined. The total coincidence rate of the kit for detecting foot-and-mouth disease inactivated vaccine immune antibodies and negative serum is as high as 95% or more, but the positive detection rate of the kit for detecting foot-and-mouth disease synthetic peptide vaccine immune antibodies is obviously improved, and the cross reaction of the kit for detecting foot-and-mouth disease virus type O vaccine and pig foot-and-mouth disease virus type O inactivated vaccine immune antibodies is effectively reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and relates to an ELISA antibody detection kit for foot-and-mouth disease virus type A as well as a preparation method and application thereof. BACKGROUND

[0002] Foot-and-mouth disease virus includes seven serotypes, and at present, O type and A type are mainly prevalent in China. The amino acid sequences of structural proteins of the two types of viruses are highly similar, so the most important difficulty in developing the foot-and-mouth disease virus structural protein antibody detection kit lies in avoiding non-specific reaction with antibodies of different serotypes, especially at present, the foot-and-mouth disease A type vaccines used in China are all combined with O type vaccines to form multivalent vaccines, including foot-and-mouth disease virus OA bivalent inactivated vaccine and foot-and-mouth disease virus OA bivalent synthetic peptide vaccine, which puts higher requirements on the detection specificity of the kit.

[0003] Through searching existing patent literatures, it is found that CN109485703A discloses a foot-and-mouth disease A type structural protein VP1 antigen epitope polypeptide and application thereof; and CN106432434A discloses an ELISA detection kit for foot-and-mouth disease A type structural protein VP1 antibody. The patent relates to a mixed polypeptide composed of one or several of sequence a (Tyr Asn Gly Thr Thr Lys Tyr Ser Thr Gly Asn Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Val Ala Ala Gln Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile Arg Ala), sequence b (Val Tyr Asn Gly Thr Ser Lys Tyr Ser Ala Pro Ala Thr Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe Asn Tyr Gly Ala Ile Arg Ala) and sequence c (Val Tyr Ser Gly Thr Ser Lys Tyr Ser Ala Ser Gln Asn Arg Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile Arg Ala), and application of the polypeptide in preparing a kit for detecting foot-and-mouth disease A type virus structural protein VP1 antibody produced after infection or immunization.

[0004] The disadvantage is that in verifying the specific reaction, the cross-reaction of the immune antibody to the synthetic peptide vaccine (polypeptide 2600+2700+2800) of swine foot-and-mouth disease O type currently used in the market is not verified. In fact, the designed polypeptide has completely consistent sequences (such as the italicized sequence) corresponding to the position of foot-and-mouth disease O type virus (such as the sequence of GH loop corresponding to foot-and-mouth disease O type virus Re-O / MYA98 / JSCZ / 2013 strain is Val Tyr Asn Gly Lys Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro Thr Ser Phe Asn Tyr Gly Ala lie Lys Ala ) especially involving the N-terminal and C-terminal sequences. In fact, the experiment of the present application found that the polypeptide sequences a (corresponding to PAF7204 in the specification of the present application), sequence b (corresponding to PWH0904 in the specification of the present application), sequence c (corresponding to PMM1304 in the specification of the present application) selected by the present application can indeed non-specifically bind to the antibody of the synthetic peptide vaccine of swine foot-and-mouth disease O type, which will lead to false positive reaction in clinical application and hinder the clinical evaluation of the real immune effect of the vaccine. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a foot-and-mouth disease virus A type ELISA antibody detection kit and its preparation method and application. The present application determines the sequence of the GH loop position of foot-and-mouth disease A type virus which non-specifically binds to the antibody of the synthetic peptide vaccine of swine foot-and-mouth disease O type and the inactivated vaccine of swine foot-and-mouth disease O type, and effectively solves the problem of non-specific binding through sequence reduction. However, the detection sensitivity of A type antibody is significantly reduced after reduction, so the detection sensitivity is further improved by displaying the polypeptide in the form of a multi-antigen peptide. Finally, the polypeptide epitope combination, the antigen coated plate containing the polypeptide epitope combination and the ELISA antibody detection kit are determined.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The present application provides a foot-and-mouth disease virus A type epitope polypeptide, and the amino acid sequence of the polypeptide is shown in sequence 1, sequence 1: (VYNGTSKYSAPATRRGDLGSLAARLAAQLPASGSS) 2K.

[0008] The present application also provides a foot-and-mouth disease virus A type epitope polypeptide, and the amino acid sequence of the polypeptide is shown in sequence 2, sequence 2: (VYSGTSKYSAPQNRRGDSGPLAARLAAQLPASGSS) 2K.

[0009] Also provided is a polypeptide of epitope of foot-and-mouth disease virus type A, the amino acid sequence of the polypeptide is shown in sequence 3: (VYNGTTKYSTGNAGRRGDLGSLAARVAAQLPASGSS) 2K.

[0010] The application also provides an antigen-coated plate, the coated plate contains three epitope polypeptides, the amino acid sequences of the polypeptides are as follows:

[0011] Sequence 1: (VYNGTSKYSAPATRRGDLGSLAARLAAQLPASGSS) 2K;

[0012] Sequence 2: (VYSGTSKYSAPQNRRGDSGPLAARLAAQLPASGSS) 2K;

[0013] Sequence 3: (VYNGTTKYSTGNAGRRGDLGSLAARVAAQLPASGSS) 2K.

[0014] The application also provides a preparation method of the antigen-coated plate, the method comprises the following steps:

[0015] S1, respectively, using solid-phase synthesis method to synthesize the epitope polypeptide;

[0016] S2, mixing the three epitope polypeptides, diluting and then adding into enzyme-linked reaction plate holes for coating, blocking, drying treatment, thus obtaining the antigen-coated plate.

[0017] As an embodiment, in step S1, the synthesis of sequence 1 comprises the following steps:

[0018] 1) Synthesizing a fully-protected peptide segment 1:

[0019] Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-OH (protected peptide segment 1);

[0020] 2) Synthesizing a protected peptide segment 2:

[0021] Fmoc-Ala-Thr(tBu)-Arg(Pbf)-Arg(Pbf)-Gly-Asp(OtBu)-Leu-Gly-Ser(tBu)-Leu-Ala-OH (protected peptide segment 2);

[0022] 3) Synthesizing a protected peptide segment 3:

[0023] H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-Lys(H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-)-Rink amide MBHA resin (protected peptide resin fragment 3);

[0024] 4) In the NMP solution of the protected peptide fragment 3, add the activated fragment of the protected fragment 2 / HOBt / DIC to carry out the coupling reaction. After the reaction is complete (Kaiser test negative), remove the Fmoc on the peptide resin with piperidine, and after washing with NMP, add the activated fragment of the protected fragment 1 / PyBOP / DIPEA to carry out the coupling reaction. After the reaction is complete (Kaiser test negative), remove the Fmoc on the peptide resin with piperidine to obtain the fully protected sequence 1 peptide resin.

[0025] 5) Remove the solid phase carrier and the amino acid side chain protection group in the sequence 1 peptide resin, and precipitate to obtain a crude peptide sample of the complete polypeptide (VYNGTSKYSAPATRRGDLGSLAARLAAQLPASGSS) 2K; purify to obtain the complete sequence 1 polypeptide.

[0026] Preferably, step 1) comprises: first synthesizing a peptide resin:

[0027] Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-2-chlorotrityl resin; remove the solid phase carrier to obtain a fully protected peptide fragment.

[0028] The synthetic peptide resin is specifically: using 2-chlorotrityl resin as a solid phase carrier, diisopropyl ethyl amine (DIPEA) as a catalyst, Fmoc-Pro-OH is connected to the resin to form Fmoc-Pro-2-chlorotrityl resin, and then 25% piperidine (PIP) / NMP is used to remove Fmoc to form H-Pro-2-chlorotrityl resin, after NMP washing to remove residual piperidine, Fmoc-Ala-OH / PyBOP / DIPEA activated amino acid coupling is added to form Fmoc-Ala-Pro-2-chlorotrityl resin, after NMP washing of residual amino acid, Fmoc is removed again, and the coupling, washing, and Fmoc removal steps are cycled, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH are sequentially coupled, and finally Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-2-chlorotrityl resin is formed.

[0029] Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-2-chlorotrityl resin.

[0030] In step 1), the peptide resin is treated with 1% trifluoroacetic acid (TFA) / dichloromethane (DCM) to remove the solid phase carrier, and the fully protected peptide segment is obtained.

[0031] Preferably, step 3) is specifically: using Rink amide MBHA resin as a solid phase carrier, adding Fmoc-Lys(Fmoc)-OH / HOBt / DIC for activated amino acid coupling to form Fmoc-Lys(Fmoc)-Rink amide MBHA resin, adding 25% PIP / NMP to remove Fmoc after NMP washing, to obtain H-Lys(ε-NH2)-Rink amide MBHA resin, NMP washing of residual piperidine, according to the sequence of the peptide segment, coupling, washing, Fmoc removal, and washing steps, and finally forming:

[0032] H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-Lys(H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-)-Rink amide MBHA resin (protective peptide resin fragment 3).

[0033] Preferably, step 5) specifically involves: adding TFA / TIS / DTT / H2O (90:5:2:3) to the fully protected sequence 1 peptide resin to remove the solid support and amino acid side chain protecting groups, and precipitating with tert-butyl methyl ether (MTBE) to obtain a crude peptide sample of the complete polypeptide (VYNGTSKYSAPATRRGDLGSLAARLAAQLPASGSS)2K.

[0034] The purification described in step 5) is as follows: the crude peptide sample is dissolved in pure water and purified by high-performance liquid chromatography (HPLC)-C18 column to obtain a complete sequence peptide with a final purity of greater than 85%.

[0035] As one implementation scheme, in step S1, the synthesis of sequence 2 includes the following steps:

[0036] 1) Fully protected peptide 4 was synthesized:

[0037] Fmoc-Val-Tyr(tBu)-Ser(tBu)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-OH (protective peptide fragment 4);

[0038] 2) Synthesis of protective peptide 5:

[0039] Fmoc-Gln(Trt)-Asn(Trt)-Arg(Pbf)-Arg(Pbf)-Gly-Asp(OtBu)-Ser(tBu)-Gly-Pro-Leu-Ala-OH (protective peptide fragment 5);

[0040] 3) Synthesize protective peptide resin fragment 3:

[0041] H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-Lys(H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-)-Rink amide MBHA resin (protected peptide resin fragment 3);

[0042] 4) In the NMP solution of the protected peptide resin fragment 3, the activated fragment of the protected fragment 5 / HOAt / DIC was added for coupling reaction. After the reaction was completed (Kaiser test negative), Fmoc on the peptide resin was removed with piperidine, and after NMP washing, the activated fragment of the protected fragment 4 / TBTU / DIPEA was added for coupling reaction. After the reaction was completed (Kaiser test negative), Fmoc on the peptide resin was removed with piperidine to obtain the fully protected sequence 2 peptide resin.

[0043] 5) The solid phase carrier and the amino acid side chain protecting group were removed in the sequence 2 peptide resin, and the complete (VYSGTSKYSAPQNRRGDSGPLAARLAAQLPASGSS) 2K crude peptide sample was obtained by precipitation. The sequence 2 polypeptide was obtained by purification.

[0044] Step 1) is specifically to synthesize a peptide resin:

[0045] Fmoc-Val-Tyr(tBu)-Ser(tBu)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-2-chlorotrityl resin; the solid phase carrier was removed to obtain the fully protected peptide segment 1.

[0046] The synthetic peptide resin is specifically: using 2-chlorotrityl resin as a solid phase carrier, diisopropyl ethyl amine (DIPEA) as a catalyst, Fmoc-Pro-OH is connected to the resin to form Fmoc-Pro-2-chlorotrityl resin, and H-Pro-2-chlorotrityl resin is formed by washing and removing Fmoc; Fmoc-Ala-OH / HBTU / DIPEA activated amino acid coupling is added to form Fmoc-Ala-Pro-2-chlorotrityl resin, and Fmoc is removed by washing and removing Fmoc, and the Fmoc is removed by washing and removing Fmoc. The steps of cyclic coupling, washing, and removing Fmoc are sequentially coupled with Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, and finally Fmoc-Val-Tyr(tBu)-Ser(tBu)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-2-chlorotrityl resin is formed.

[0047] The peptide resin is treated with 1% trifluoroacetic acid (TFA) / dichloromethane (DCM) to remove the solid phase carrier, and the fully protected peptide segment 4 is obtained.

[0048] Preferably, step 3) is specifically: using Rink amide MBHA resin as a solid phase carrier, adding Fmoc-Lys(Fmoc)-OH / HOAt / EDC.HCl as an activated amino acid to form Fmoc-Lys(Fmoc)-Rink amide MBHA resin, and then adding 25% PIP / NMP to remove Fmoc after NMP washing to obtain H-Lys(ε-NH2)-Rink amide MBHA resin, NMP washing residual piperidine, and coupling, washing, removing Fmoc and washing steps according to the sequence of the peptide segment, and finally forming the protected peptide resin segment 3.

[0049] Preferably, in step 5), the purification is: dissolving the crude peptide sample in pure water, purifying through a high-pressure liquid chromatography (HPLC)-C18 chromatographic column to obtain a complete sequence 2 polypeptide with a final purity of more than 85%.

[0050] Preferably, in step 5), TFA / TIS / EDT / H2O (90:5:3:2) is added to the peptide resin of sequence 2 to remove the solid support and the side chain protecting groups of the amino acids, and the crude peptide sample of intact (VYSGTSKYSAPQNRRGDSGPLAARLAAQLPASGSS)2K is obtained by precipitation with tert-butyl methyl ether (MTBE).

[0051] As an embodiment, in step S1, the synthesis of sequence 3 comprises the following steps:

[0052] 1) Synthesis of the fully protected peptide segment 6:

[0053] Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Thr(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Thr(tBu)-Gly-OH (protected peptide segment 6);

[0054] 2) Synthesis of the protected peptide segment 7:

[0055] Fmoc-Asn(Trt)-Ala-Gly-Arg(Pbf)-Arg(Pbf)-Gly-Asp(OtBu)-Leu-Gly-Ser(tBu)-Leu-Ala-OH (protected peptide segment 7);

[0056] 3) Synthesis of the protected peptide resin segment 8:

[0057] H-Ala-Arg(Pbf)-Val-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-Lys(H-Ala-Arg(Pbf)-Val-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-)-Rink amide MBHA resin (protected peptide resin segment 8);

[0058] 4) In the NMP solution of the protected peptide resin segment 8, the activated segment of protected segment 7 / HOBt / DIC is added for coupling reaction, after the reaction is complete (Kaiser test negative), piperidine is added to remove Fmoc on the peptide resin, after NMP washing, the activated segment of protected segment 6 / HATU / DIPEA is added for coupling reaction, after the reaction is complete (Kaiser test negative), piperidine is added to remove Fmoc on the peptide resin, to obtain the fully protected peptide resin of sequence 3;

[0059] 5) removing the solid support and the side chain protecting groups of the amino acids from the sequence 3 peptide resin, and precipitating to obtain a crude peptide sample of (VYNGTTKYSTGNAGRRGDLGSLAARVAAQLPASGSS) 2K; purifying to obtain the sequence 3 polypeptide.

[0060] In step 1), the peptide resin is synthesized as follows:

[0061] Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Thr(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Thr(tBu)-Gly-2-chlorotrityl resin; removing the solid support to obtain the fully protected peptide segment 6.

[0062] The synthetic peptide resin is specifically as follows:

[0063] With 2-chlorotrityl resin as the solid support and diisopropyl ethyl amine (DIPEA) as the catalyst, Fmoc-Gly-OH is connected to the resin to form Fmoc-Gly-2-chlorotrityl resin, and then 25% piperidine (PIP) / NMP is used to remove Fmoc to form H-Gly-2-chlorotrityl resin. After NMP washing to remove residual piperidine, Fmoc-Thr(tBu)-OH / PyBOP / DIPEA activated amino acid is coupled to form Fmoc-Thr(tBu)-Gly-2-chlorotrityl resin. After NMP washing to remove residual amino acid, Fmoc is removed again. The coupling, washing, and Fmoc removal steps are repeated in a cycle, and Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH are sequentially coupled, to finally form Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Thr(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Thr(tBu)-Gly-2-chlorotrityl resin.

[0064] The peptide resin is treated with trifluoroethanol (TFE) / acetic acid / dichloromethane (DCM) (1:1:8) to remove the solid support, to obtain the fully protected peptide segment 6.

[0065] Preferably, step 3) is specifically: taking Rink amide MBHA resin as a solid carrier, adding Fmoc-Lys(Fmoc)-OH / HOBt / DIC as an activated amino acid to couple to form Fmoc-Lys(Fmoc)-Rink amide MBHA resin, washing with NMP, then adding 25% PIP / NMP to remove Fmoc, to obtain H-Lys(ε-NH2)-Rink amide MBHA resin, washing the residual piperidine, and coupling, washing, removing Fmoc and washing according to the sequence of the peptide segment, to finally form the protected peptide resin segment 8.

[0066] Preferably, step 5) is specifically: adding TFA / TIS / DTT / H2O (90:5:4:1) to the fully protected sequence 3 peptide resin to remove the solid carrier and the amino acid side chain protecting group, and precipitating with tert-butyl methyl ether (MTBE) to obtain a crude peptide sample of complete (VYNGTTKYSTGNAGRRGDLGSLAARVAAQLPASGSS) 2K.

[0067] Preferably, in step 5), the purification is dissolving the crude peptide sample in pure water, and purifying through a high-pressure liquid chromatography (HPLC)-C18 chromatography column to obtain a complete sequence 3 polypeptide with a final purity of more than 85%.

[0068] As an embodiment, in step S2, the mixing mass ratio of the epitope polypeptides of sequence 1, sequence 2 and sequence 3 is 1:1:1 to 1:3:3, or 1:1:1 to 3:1:3, or 1:1:1 to 3:3:1. Preferably, the three epitope polypeptides are mixed in equal amounts.

[0069] As an embodiment, in step S2, the coating is performed at 2-8°C for 18-24 hours.

[0070] As an embodiment, in step S2, the coating solution is a carbonate-bicarbonate buffer with a pH value of 9.55-9.65, containing 0.02%-0.08% DMSO and 0.3%-1% Tween20.

[0071] The application also provides a foot-and-mouth disease virus type A ELISA antibody detection kit, which comprises the antigen-coated plate as described above.

[0072] As an embodiment, the kit further comprises an enzyme conjugate, an enzyme conjugate diluent, a sample diluent, a color developing solution, a termination solution, and a 25-fold washing solution.

[0073] The application also provides a use of the aforementioned antigen-coated plate in the preparation of an ELISA antibody detection kit for foot-and-mouth disease virus type A. The kit is used to detect the level of foot-and-mouth disease type A antibodies. Further, the kit is used to detect type A antibodies in the serum of animals after the action of foot-and-mouth disease type A virus and vaccine. In some embodiments, the kit is used to detect type A antibodies in the serum of animals after the use of foot-and-mouth disease type A vaccine and type O vaccine in multiple valences.

[0074] The long-term prevalent foot-and-mouth disease virus in China is type O, which is the most serious, and the dominant prevalent strain is SEA topology Mya-98 strain. Since 2009, foot-and-mouth disease virus type A has begun to prevail in China, and the representative strains include A / Sea-97 G1 branch virus A / HuBWH / CHA / 2009 strain and A / Sea-97 G2 branch virus A / GDMM13 / CHA / 2013 strain. At this time, the OA bivalent inactivated vaccine and synthetic peptide vaccine for preventing and controlling foot-and-mouth disease of type A and type O viruses at the same time play a key role in epidemic prevention and control, including the pig foot-and-mouth disease O type, A type bivalent inactivated vaccine (Re / O / MYA98 / JSCZ / 2013 strain + Re-A / WH / 09 strain) of Shenglian Biomedicine (Shanghai) Co., Ltd. Lanzhou Branch, pig foot-and-mouth disease O type, A type bivalent synthetic peptide vaccine (polypeptide 2700+2800+MM13), etc. The antigens of the inactivated vaccine are artificially cultured and chemically inactivated viruses, and the antigens of the synthetic peptide vaccine contain the key neutralizing epitope GH loop sequence of the representative strains such as O type MYA-98 strain and A type A / GDMM13 / CHA / 2013 strain. The vaccine can produce very high concentration of neutralizing antibodies against the GH loop, so it can efficiently prevent O type and A type foot-and-mouth disease virus infection at the same time. According to the National Animal Disease Immunization Technology Guide in 2022, the method specified in GB / T 18935-2018 "Foot-and-mouth disease diagnosis technology" is required for routine detection of foot-and-mouth disease vaccine antibodies. For inactivated vaccine immunization, liquid-phase blocking ELISA and solid-phase competitive ELISA are used to detect immune antibodies; for synthetic peptide vaccine immunization, VP1 structural protein ELISA is used to detect immune antibodies. However, because the amino acid sequences of O type virus and A type virus structural proteins have a similarity of more than 75%, and the amino acid sequences of the GH loop located on the structural protein are also highly similar, especially the N-terminal and C-terminal sequences are completely identical, therefore, in the presence of very high concentration of specific antibodies, avoiding non-specific detection of O type antibodies becomes the most important difficulty in the development of A type VP1 structural protein ELISA antibody detection kit.

[0075] Designing polypeptides as antigens for ELISA antibody detection may effectively reduce non-specific reactions, but the difficulty of directly applying polypeptide antigens to ELISA antibody detection lies in that each amino acid residue on the polypeptide can be randomly combined with the surface of the 96-well enzyme-linked reaction plate by various unpredictable forces, including hydrophilic force, hydrophobic force, van der Waals force or ionic bond, etc., which causes the polypeptide to lie on the plate hole surface and cannot be fully displayed in the solution, which is not conducive to the combination with the antibody, resulting in reduced diagnostic sensitivity (as described in the background of patent CN102998460B).

[0076] Multimeric antigen peptides are formed by amino and carboxyl condensation reactions on the alpha and epsilon amino groups of lysine to form dendritic peptide molecules containing multiple polypeptides, in order to achieve the purpose of displaying polypeptides. However, as the number of polypeptides increases and the number of amino acid residues in the polypeptides increases, the mutual entanglement between the peptide chains and the mutual entanglement between the peptide chains and the matrix resin will limit the interaction between the polypeptides and the antibodies, so the number of amino acid residues contained in the multimeric antigen peptides reported for diagnostic purposes is generally not more than 25 (according to the literature DOI 10.1007 / s13337-013-0162-z), and the preparation and application of super-long multimeric antigen peptides (such as the polypeptides in the present application containing 35 amino acid residues) for diagnostic purposes are industry difficulties.

[0077] Compared with the prior art, the present application has the following beneficial effects:

[0078] 1) The present application determines the sequence of the foot-and-mouth disease type A virus GH loop epitope polypeptide that is non-specifically combined with the foot-and-mouth disease type O synthetic peptide vaccine antibody and the foot-and-mouth disease type O inactivated vaccine antibody, effectively solves the problem of non-specific combination through sequence reduction; however, the sensitivity of the reduced polypeptide (containing 32 amino acid residues) for A-type antibody detection is significantly reduced, and a super-long multimeric antigen peptide (containing 35 amino acid residues per branch peptide) is prepared by an optimized solid-phase synthesis method, and an antigen-coated plate containing the super-long multimeric antigen peptide is prepared by optimizing the components of the polypeptide coating solution, which significantly improves the sensitivity of antibody detection, and is the first successful preparation of a super-long multimeric antigen peptide and its application in antibody detection.

[0079] 2) The present application finally determines the polypeptide epitope combination, the antigen-coated plate containing the polypeptide epitope combination, and the ELISA antibody detection kit; compared with the commercial liquid-phase blocking kit and the competitive kit, the total coincidence rate of the present kit for foot-and-mouth disease inactivated vaccine immune antibody and negative serum detection is as high as 95% or more, but the positive detection rate of foot-and-mouth disease synthetic peptide vaccine immune antibody is significantly improved.

[0080] 3) Compared with the commercialized pig foot-and-mouth disease type A VP1 structural protein ELISA antibody detection kit, the kit can effectively reduce the cross reaction of the immune antibody to the foot-and-mouth disease virus O type vaccine (pig foot-and-mouth disease O type synthetic peptide vaccine (polypeptide 2600+2700+2800), pig foot-and-mouth disease virus O type inactivated vaccine (Re-O / MYA98 / JSCZ / 2013 strain));

[0081] 4) The kit of the present application is beneficial to the real clinical monitoring of the immune effect of the foot-and-mouth disease type A vaccine, especially the type A synthetic peptide vaccine. DETAILED DESCRIPTION

[0082] The present application will be described in detail below in combination with specific embodiments. The following embodiments will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be pointed out that, for those skilled in the art, several changes and improvements can be made without departing from the concept of the present application. These all belong to the protection scope of the present application.

[0083] Example 1, design, preparation and screening of polypeptide antigen

[0084] The key neutralizing B cell epitope GH loop (amino acid 129-171) was selected when the foot-and-mouth disease synthetic peptide vaccine was designed, and the GH loop corresponding amino acid sequences of the corresponding strain sequences were downloaded from NCBI in combination with the domestic foot-and-mouth disease type A virus main epidemic strain (A / GDMM / 2013 strain) and vaccine strain (Re-A / WH / 09 strain and AF72 strain), and each candidate antigen polypeptide was designed (see Table 1).

[0085] Table 1 candidate epitope polypeptide

[0086]

[0087] The polypeptides are synthesized by Fmoc solid-phase synthesis method, and the synthesis sequence is from the C-terminal to the N-terminal. Among them, the polyantigen peptide, because the polypeptide is longer and contains 35 amino acid residues, cannot be obtained by the conventional linear synthesis method. The polypeptide synthesis reaction is terminated at the 16th residue L, which leads to the inability to prepare a complete antigen. After many optimizations, the segmented synthesis method is used to solve this problem. Specifically, PWH09-5 is divided into three fragments (VYNGTSKYSAP, ATRRGDLGSLA and (ARLAAQLPASGSS) 2K) for synthesis, and the three polypeptides are synthesized separately to solve the problem that the 16th L residue cannot be synthesized directly. First, ARLAAQLPASGSS is connected to the N-terminal amino group and the side chain amino group of Lys to form a binary branched peptide (ARLAAQLPASGSS) 2K, and then the polypeptides ATRRGDLGSLA and VYNGTSKYSAP are sequentially spliced to the N-terminus of (ARLAAQLPASGSS) 2K to obtain the complete polypeptide PWH09-5. Similarly, PMM13-2 is divided into three fragments (VYSGTSKYSAP, QNRRGDSGPLA and (ARLAAQLPASGSS) 2K) for synthesis, and first ARLAAQLPASGSS is connected to the N-terminal amino group and the side chain amino group of Lys to form a binary branched peptide (ARLAAQLPASGSS) 2K, and then the polypeptides QNRRGDSGPLA and VYSGTSKYSAP are sequentially spliced to the N-terminus of (ARLAAQLPASGSS) 2K to obtain the complete polypeptide PMM13-2. Similarly, PAF72-2 is divided into three fragments (VYNGTTKYSTG, NAGRRGDLGSLA and (ARVAAQLPASGSS) 2K) for synthesis, and first ARVAAQLPASGSS is connected to the N-terminal amino group and the side chain amino group of Lys to form a binary branched peptide (ARVAAQLPASGSS) 2K, and then the polypeptides NAGRRGDLGSLA and VYNGTTKYSTG are sequentially spliced to the N-terminus of (ARVAAQLPASGSS) 2K to obtain the complete polypeptide PAF72-2.

[0088] Specifically, the synthesis of sequence 1 includes the following steps:

[0089] 1) Fmoc-Pro-OH was linked to the resin to form Fmoc-Pro-2-chlorotrityl resin using 2-chlorotrityl resin as the solid support and diisopropylethylamine (DIPEA) as the catalyst, then Fmoc was removed to form H-Pro-2-chlorotrityl resin using 25% piperidine (PIP) / NMP, after NMP washing to remove residual piperidine, Fmoc-Ala-OH / PyBOP / DIPEA was added to activate the amino acid for coupling to form Fmoc-Ala-Pro-2-chlorotrityl resin, after NMP washing to remove residual amino acid, Fmoc was removed again, and the coupling, washing, and Fmoc removal steps were repeated to sequentially couple Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, finally forming Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-2-chlorotrityl resin.

[0090] 2) The peptide resin from step 1 was treated with 1% trifluoroacetic acid (TFA) / dichloromethane (DCM) to remove the solid support, obtaining the fully protected peptide segment Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-OH (protected peptide segment 1).

[0091] 3) The protected peptide segment Fmoc-Ala-Thr(tBu)-Arg(Pbf)-Arg(Pbf)-Gly-Asp(OtBu)-Leu-Gly-Ser(tBu)-Leu-Ala-OH (protected peptide segment 2) was also obtained using the method of steps 1 and 2.

[0092] 4) Fmoc-Lys(Fmoc)-Rink amide MBHA resin was formed by adding Fmoc-Lys(Fmoc)-OH / HOBt / DIC as activated amino acid coupling with Rink amide MBHA resin as solid support, and then 25% PIP / NMP was added to remove Fmoc after NMP washing, to obtain H-Lys(ε-NH2)-Rink amide MBHA resin, NMP was used to wash the residual piperidine, and the coupling, washing, Fmoc removal and washing steps were performed according to the sequence of the peptide segment, to finally form H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-Lys(H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-)-Rink amide MBHA resin (protected peptide resin segment 3).

[0093] 5) In the NMP solution of the protected peptide resin segment 3, the coupling reaction was performed by adding the activated segment of protective segment 2 / HOBt / DIC, and after the reaction was complete (Kaiser test negative), piperidine was used to remove Fmoc on the peptide resin, and then the activated segment of protective segment 1 / PyBOP / DIPEA was added for coupling reaction after NMP washing, and after the reaction was complete (Kaiser test negative), piperidine was used to remove Fmoc on the peptide resin, to obtain the fully protected sequence 1 peptide resin.

[0094] 6) In the fully protected sequence 1 peptide resin, TFA / TIS / DTT / H2O (90:5:2:3) was added to remove the solid support and amino acid side chain protecting groups, and tert-butyl methyl ether (MTBE) was used for precipitation to obtain the crude peptide sample of the complete polypeptide (VYNGTSKYSAPATRRGDLGSLAARLAAQLPASGSS) 2K.

[0095] 7) The crude peptide sample of step 6 was dissolved in pure water, and purified by high pressure liquid chromatography (HPLC)-C18 chromatography column to obtain the complete sequence 1 polypeptide with a final purity of more than 85%.

[0096] The synthesis of sequence 2 includes the following steps:

[0097] 1) Fmoc-Pro-OH was linked to the resin to form Fmoc-Pro-2-chlorotrityl resin using 2-chlorotrityl resin as the solid support and diisopropylethylamine (DIPEA) as the catalyst, then Fmoc was removed to form H-Pro-2-chlorotrityl resin using 25% piperidine (PIP) / NMP, after NMP washing to remove residual piperidine, Fmoc-Ala-OH / HBTU / DIPEA activated amino acid was added for coupling to form Fmoc-Ala-Pro-2-chlorotrityl resin, after NMP washing to remove residual amino acid, Fmoc was removed again, and the coupling, washing, and Fmoc removal steps were repeated to sequentially couple Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, finally forming Fmoc-Val-Tyr(tBu)-Ser(tBu)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-2-chlorotrityl resin.

[0098] 2) The peptide resin described above was treated with 1% trifluoroacetic acid (TFA) / dichloromethane (DCM) to remove the solid support, obtaining the fully protected peptide segment Fmoc-Val-Tyr(tBu)-Ser(tBu)-Gly-Thr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Ala-Pro-OH (protected peptide segment 4).

[0099] 3) The protected peptide segment Fmoc-Gln(Trt)-Asn(Trt)-Arg(Pbf)-Arg(Pbf)-Gly-Asp(OtBu)-Ser(tBu)-Gly-Pro-Leu-Ala-OH (protected peptide segment 5) was also obtained by the method of steps 1 and 2.

[0100] 4) Fmoc-Lys(Fmoc)-OH / HOAt / EDC.HCl as activated amino acid coupling to form Fmoc-Lys(Fmoc)-Rink amide MBHA resin, after NMP washing, 25% PIP / NMP was added to remove Fmoc, and H-Lys(ε-NH2)-Rink amide MBHA resin was obtained, NMP was used to wash the residual piperidine, and the coupling, washing, Fmoc removal and washing steps were performed according to the sequence of the peptide segment, and finally H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-Lys(H-Ala-Arg(Pbf)-Leu-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-)-Rink amide MBHA resin (protected peptide resin fragment 3) was formed.

[0101] 5) In the NMP solution of the protected peptide resin fragment 3, the activated fragment of the protective fragment 5 / HOAt / DIC was added for coupling reaction, after the reaction was complete (Kaiser test negative), piperidine was used to remove Fmoc on the peptide resin, after NMP washing, the activated fragment of the protective fragment 4 / TBTU / DIPEA was added for coupling reaction, after the reaction was complete (Kaiser test negative), piperidine was used to remove Fmoc on the peptide resin, and the fully protected sequence 2 peptide resin was obtained.

[0102] 6) TFA / TIS / EDT / H2O (90:5:3:2) was added to the fully protected sequence 2 peptide resin to remove the solid phase carrier and the amino acid side chain protecting group, and tert-butyl methyl ether (MTBE) was used for precipitation to obtain a crude peptide sample of complete (VYSGTSKYSAPQNRRGDSGPLAARLAAQLPASGSS) 2K;.

[0103] 7) The crude peptide sample of step 6 was dissolved in pure water, and purified by high pressure liquid chromatography (HPLC)-C18 chromatographic column to obtain a complete sequence 2 polypeptide with a final purity of more than 85%.

[0104] The synthesis of sequence 3 includes the following steps:

[0105] 1) Fmoc-Gly-OH was linked to the resin to form Fmoc-Gly-2-chlorotrityl resin using 2-chlorotrityl resin as the solid support and diisopropylethylamine (DIPEA) as the catalyst, then Fmoc was removed to form H-Gly-2-chlorotrityl resin using 25% piperidine (PIP) / NMP, after NMP washing to remove residual piperidine, Fmoc-Thr(tBu)-OH / PyBOP / DIPEA activated amino acid was added for coupling to form Fmoc-Thr(tBu)-Gly-2-chlorotrityl resin, after NMP washing to remove residual amino acid, Fmoc was removed again, and the coupling, washing, and Fmoc removal and washing steps were repeated to sequentially couple Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH, to finally form Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Thr(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Thr(tBu)-Gly-2-chlorotrityl resin.

[0106] 2) The peptide resin described above was treated with trifluoroethanol (TFE) / acetic acid / dichloromethane (DCM) (1:1:8) to remove the solid support, to obtain the fully protected peptide segment Fmoc-Val-Tyr(tBu)-Asn(Trt)-Gly-Thr(tBu)-Thr(tBu)-Lys(Boc)-Tyr(tBu)-Ser(tBu)-Thr(tBu)-Gly-OH (protected peptide segment 6).

[0107] 3) The protected peptide segment Fmoc-Asn(Trt)-Ala-Gly-Arg(Pbf)-Arg(Pbf)-Gly-Asp(OtBu)-Leu-Gly-Ser(tBu)-Leu-Ala-OH (protected peptide segment 7) was also obtained by the method of steps 1 and 2.

[0108] 4) Fmoc-Lys(Fmoc)-Rink amide MBHA resin was formed by adding Fmoc-Lys(Fmoc)-OH / HOBt / DIC as activated amino acid coupling with Rink amide MBHA resin as solid phase carrier, and then 25% PIP / NMP was added to remove Fmoc after NMP washing, to obtain H-Lys(ε-NH2)-Rink amide MBHA resin, NMP was used to wash the residual piperidine, and the coupling, washing, Fmoc removal and washing steps were performed according to the sequence of the peptide segment, and finally H-Ala-Arg(Pbf)-Val-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-Lys(H-Ala-Arg(Pbf)-Val-Ala-Ala-Gln(Trt)-Leu-Pro-Ala-Ser(tBu)-Gly-Ser(tBu)-Ser(tBu)-)-Rink amide MBHA resin (protected peptide resin segment 8) was formed.

[0109] 5) In the NMP solution of the protected peptide resin segment 8, the coupling reaction was performed by adding the activated segment of protective segment 7 / HOBt / DIC, and after the reaction was complete (Kaiser test negative), piperidine was used to remove Fmoc on the peptide resin, and then the coupling reaction was performed by adding the activated segment of protective segment 6 / HATU / DIPEA after NMP washing, and after the reaction was complete (Kaiser test negative), piperidine was used to remove Fmoc on the peptide resin, to obtain the fully protected sequence 3 peptide resin.

[0110] 6) In the fully protected sequence 3 peptide resin, TFA / TIS / DTT / H2O (90:5:4:1) was added to remove the solid phase carrier and the amino acid side chain protecting group, and tert-butyl methyl ether (MTBE) was used for precipitation to obtain a crude peptide sample of complete (VYNGTTKYSTGNAGRRGDLGSLAARVAAQLPASGSS) 2K.

[0111] 7) The crude peptide sample of step 6 was dissolved in pure water, and high pressure liquid chromatography (HPLC) was used to purify the C18 chromatographic column to obtain the final sequence 3 polypeptide with a purity of more than 85%.

[0112] Further, the optimal polypeptide is screened by detecting the serum of pigs immunized with foot-and-mouth disease virus type A Re-A / WH / 09 inactivated vaccine (No. SA1), the serum of pigs immunized with foot-and-mouth disease virus type A A / GDMM13 / CHA / 2013 inactivated vaccine (No. SA2), the serum of pigs immunized with foot-and-mouth disease virus type A AF72 inactivated vaccine (No. SA3), the serum of pigs immunized with foot-and-mouth disease virus type O synthetic peptide vaccine (polypeptides 2600+2700+2800) (No. SO), and the serum of pigs negative to foot-and-mouth disease virus antibody (No. SN), and the optimal polypeptide should have high sensitivity and high specificity.

[0113] It is found in the present study that the detection ability of the polypeptide coated with the coating solution of carbonate-bicarbonate buffer (0.1 M, pH 9.4) described in the literature (DOI: 10.1016 / s0022-1759(01)00444-6) is even weaker than the detection effect of the single polypeptide PWH0903 (see Table 2), which may be because the super-long polypeptide is directly used, and the polypeptide chains are inevitably intertwined with each other, so that the polypeptide cannot be fully displayed in the solution. Therefore, the components of the polypeptide coating solution are optimized, and the preferred coating solution of carbonate-bicarbonate buffer (0.1 M, pH 9.55-9.65, containing 0.02%-0.08% DMSO and 0.3%-1% Tween20) is obtained through multiple conditions screening. As shown in Table 2, the antigen-coated plate prepared by using the coating solution of the present application can significantly improve the detection value of sample SA1, indicating that the super-long polypeptide needs specific coating and display conditions to achieve the intended purpose.

[0114] In one case of the present application, the polypeptide coating method is to dilute the polypeptide to 1 μg / ml with carbonate-bicarbonate buffer (0.1 mol / L, pH 9.55-9.65, containing 0.03% DMSO and 0.5% TWEEN20), add 100 μl to each well of a 96-well enzyme-linked reaction plate, and coat at 2-8°C for 18-24 hours. Take out and discard the antigen coating solution in each well. Prepare 3% BSA (90 g BSA dissolved in 3 L PBS buffer), add 250 μl to each well of the coated enzyme-linked reaction plate, and seal at room temperature for 60 minutes. Discard the blocking solution. Using the solution components of the conventional indirect ELISA method and the antigen-coated plate prepared in this step, the serum samples SA1, SA2, SA3, SO and SN to be detected are detected according to the conventional indirect ELISA method, each sample is tested 3 times and the average value is calculated, whether the OD450nm value of the detected sample SO will non-specifically detect the foot-and-mouth disease virus type O synthetic peptide vaccine is preliminarily investigated, and the OD450nm value of the detected samples SA1, SA2 and SA3 is preliminarily investigated for sensitivity. The results are shown in Tables 2-4.

[0115] According to Table 2, the sample SA1 values of the relatively complete GH loop epitope polypeptides PWH09 and PWH0904 for the strain are 1.212 and 1.112, respectively, indicating that both polypeptides can effectively bind the antibodies in the sample SA1, but the OD450nm value of the sample SO is as high as 0.828, suggesting that the relatively complete GH loop epitope polypeptide has obvious cross-reaction with the O-type synthetic peptide vaccine antibodies. Further, the partial truncation optimization of the N-terminal sequence of the antigen does not reduce the cross-reaction (see PWH09-2), but the partial truncation optimization of the C-terminal sequence of the antigen significantly reduces the cross-reaction (see PWH09-3), and the OD450nm value of the sample SO is less than 0.3, but at the same time, it is observed that the detection sensitivity of the antigen is significantly reduced, and the OD450nm value of the sample SA1 is reduced to 0.754, with a reduction of 37.8% compared with the complete polypeptide. This study shows that the construction of PWH09-3 into a binary polyantigen peptide PWH09-4, a two-branch polymeric peptide containing a linker PWH09-5, and a four-branch polymeric peptide PWH09-6 can significantly improve the detection sensitivity, and the OD450nm values of the sample SA1 are increased to 2.112, 2.714, and 3.122, respectively. The above results show that PWH09-3 already contains sufficient polypeptide sequences for antibody detection, but direct application of the polypeptide to the bottom of the coated plate is not conducive to antibody binding, and the construction of a two-branch or four-branch polymeric antigen peptide can improve the antibody binding capacity, and the specific mechanism is not clear, which may be related to the fact that it is beneficial to the display of the polypeptide in the solution, increasing the probability of antibody binding in the solution, and increasing the flexible linker can further enhance this effect, in addition, the four-branch polymeric antigen peptide is superior to the two-branch polymeric antigen peptide. The polymeric antigen peptide is constructed using lysine as the core, and the amino and carboxyl condensation reactions are carried out on the alpha and epsilon amino groups of lysine, respectively, to form a dendritic peptide molecule containing multiple polypeptides. This reaction is complex, involving high-precision interactions between multiple peptide chains and between the peptide chains and the substrate resin, and the more the number of polypeptides increases, the more difficult the synthesis becomes. In actual preparation, the synthesis yield of the four-branch polymeric antigen peptide PWH09-6 is only 45.7%-50.2%, and the purity is only 31.2%-44.7%, while the synthesis yield of the two-branch polymeric antigen peptide PWH09-5 can reach 76.8%-85.1%, and the purity can reach 75.4%-83.7%. Therefore, from the perspective of controlling the process cost, PWH09-5 is selected as the optimal polypeptide for the strain, which contains two polypeptides against the A-type foot-and-mouth disease virus Re-A / WH / 09 strain, and the two polypeptides are connected to the alpha and epsilon amino groups of lysine through the C-terminal GSS linker, respectively, to form a two-branch polymeric antigen peptide.

[0116] According to Table 3, the OD450nm values of sample SA2 detected by relatively complete GH loop epitope polypeptides PMM13 and PMM1304 are 0.545 and 0.536 respectively, indicating that both polypeptides can effectively bind to the antibodies in sample SA2, but the OD450nm values of sample SO detected by the two polypeptides are as high as 0.405 and 0.401 respectively, suggesting that the relatively complete GH loop epitope polypeptide also has obvious cross-reaction with the O type synthetic peptide vaccine antibodies. The OD450nm value of sample SA2 detected by the binary branched poly-peptide PMM13-2 can be increased to 1.855, and has the highest signal-to-noise ratio S / N value of 22.622, and there is no cross-reaction in the detection of sample SO. Therefore, PMM13-2 is selected as the optimal polypeptide for the strain.

[0117] According to Table 4, the OD450nm values of sample SA3 detected by relatively complete GH loop epitope polypeptides PAF72 and PAF7204 are 0.885 and 0.755 respectively, indicating that both polypeptides can effectively bind to the antibodies in sample SA3, but the OD450nm values of sample SO detected by the two polypeptides are as high as 0.438 and 0.512 respectively, suggesting that the relatively complete GH loop epitope polypeptide also has obvious cross-reaction with the O type synthetic peptide vaccine antibodies. The OD450nm value of sample SA3 detected by the binary branched poly-peptide PAF72-2 can be increased to 2.157, and has the highest signal-to-noise ratio S / N value of 22.237, and there is no cross-reaction in the detection of sample SO. Therefore, PAF72-2 is selected as the optimal polypeptide for the strain.

[0118] Table 2 Comparison of antibody detection effects of different candidate polypeptides for A type foot-and-mouth disease virus Re-A / WH / 09 strain

[0119]

[0120] Table 3 Comparison of antibody detection effects of different candidate polypeptides for A type foot-and-mouth disease virus A / GDMM / 2013 strain

[0121]

[0122] Table 4 Comparison of antibody detection effects of different candidate polypeptides for A type foot-and-mouth disease virus AF72 strain

[0123]

[0124] To further verify whether the polypeptide combination interferes with each other and whether it can achieve broad-spectrum detection of antibodies against different strains, the candidate polypeptides were combined and used to detect each serum, and the results are shown in Table 5. PWH09-5, PMM13-2, and PAF72-2 were used alone, and only the antibodies against the corresponding strain vaccine immunization could be detected, and the cross-reactivity of the antibodies against other strain vaccines was weak, which was consistent with the characteristics of the high variation of the GH loop, and also indicated that the application of single polypeptide was limited. After the combination of the three polypeptides, each representative A-type foot-and-mouth disease vaccine immunization antibody could be well detected, and the OD450nm value was similar to the detection results of the corresponding single polypeptide, and the O-type foot-and-mouth disease vaccine immunization serum was not detected.

[0125] Table 5 Detection effect of polypeptide combination on foot-and-mouth disease antibodies

[0126]

[0127] Example 2, preparation and assembly of ELISA antibody detection kit

[0128] The specific preparation process is as follows:

[0129] (1) Preparation of polypeptide antigen

[0130] The synthesis of the polypeptide antigen was synthesized by Fmoc solid-phase synthesis method. The synthesis steps were input into the polypeptide synthesizer, and the machine automatically completed the synthesis. The synthesized peptide was freeze-dried in a freeze-drier for 3 days to form a freeze-dried product. The freeze-dried peptide was weighed, packaged in brown bottles, sealed and stored, and labeled. It was stored below -20°C. The main peak should contain more than 80% of the chromatographic integration area, that is, the purity of the polypeptide is more than 80%, which was determined by high performance liquid chromatography.

[0131] (2) Preparation of antigen-coated plate

[0132] Three kinds of polypeptide antigens were mixed in equal amounts, diluted with carbonate-bicarbonate buffer (0.1 mol / L, pH 9.6, containing 0.05% DMSO and 0.5% TWEEN20) to 1 μg / ml, and 0.1 ml was added to each well of the 96-well enzyme-linked reaction plate. Coating at 2~8°C for 18~24 hours, taking out, and discarding the antigen coating solution in the well. Use the blocking solution to inject each well of the coated enzyme-linked reaction plate, 0.33 ml per well, and block at room temperature for 30 minutes, then discard the blocking solution. Place the coated plate in a vacuum drying machine and dry at room temperature for 30 minutes. After drying, immediately place each plate with 2g of drying agent in an aluminum foil bag and seal at high temperature. The antigen-coated plate is stored at 2~8°C.

[0133] (3) Preparation of ELISA antibody detection kit

[0134] The components of the kit include antigen-coated plates, enzyme conjugate, enzyme conjugate diluent, sample diluent, color developing solution, termination solution, 25-fold washing solution, etc. Each main component is prepared according to the General Methods for Optimization and Preparation in the Veterinary Pharmacopoeia of the People's Republic of China (2020 edition) or the industry. One of the schemes is as follows:

[0135] Antigen-coated plates: prepared according to the method of step (2) of this embodiment. One or two 96-well enzyme-linked reaction plates containing the above-mentioned antigens.

[0136] Positive control serum: high-titer serum prepared by immunizing specific-pathogen-free pigs with porcine foot-and-mouth disease virus OA inactivated vaccine as immunogen, adding 1000 U / ml streptomycin and 1000 U / ml penicillin, and sterilizing with a 0.2 μm filter membrane as the positive control serum of the kit. One bottle (0.5 ml).

[0137] Negative control serum: normal pig serum without foot-and-mouth disease antibody, added with 1000 U / ml streptomycin and 1000 U / ml penicillin, sterilized with a 0.2 μm filter membrane, as the negative control serum of the kit. One tube (1.5 ml).

[0138] Enzyme conjugate: horseradish peroxidase-labeled anti-pig IgG polyclonal antibody, one bottle (0.3 ml).

[0139] Sample diluent: 0.15 M PBS buffer containing 3% (g / ml) BSA, pH 7.4, filtered with a 0.45 μm filter. One bottle (30 ml or 45 ml).

[0140] Enzyme conjugate diluent: 30 L of sterile purified water containing 60 g of anhydrous sodium dihydrogen phosphate, 4 g of disodium hydrogen phosphate dihydrate, 50 g of sodium chloride, 6 g of gentamicin, and 10 g of casein, pH 7.2, filtered with a 0.45 μm filter. One bottle (20 ml or 30 ml).

[0141] TMB substrate working solution: single-component tetramethylbenzidine (TMB) color developing solution. One bottle (15 ml or 25 ml). 25-fold washing solution: 0.25 M phosphate buffer containing 20%-30% (ml / ml) Tween-20 and 2.5% (ml / ml) ProClin 300, pH 7.4, sterilized with a 0.2 μm filter. One bottle (50 ml).

[0142] Termination solution: 2 mol / L sulfuric acid solution. One bottle (15 ml or 25 ml).

[0143] Each main component and other auxiliary components of each kit, including sample dilution plates, instructions, disposable plate sealing membranes, positioning labels, are assembled into a kit according to the following Table 6:

[0144] Table 6 ELISA kit composition

[0145]

[0146] Example 3, clinical application effect evaluation

[0147] The commercialized foot-and-mouth disease virus type A liquid-phase blocking ELISA antibody detection kit and the commercialized foot-and-mouth disease virus type A antibody ELISA kit (solid-phase blocking ELISA method) reported in the literature (Kit selection and evaluation for detection of antibody level of synthetic peptide vaccine of foot-and-mouth disease virus of pigs) were used respectively to detect 368 pig sera, and the coincidence rate was compared. The 368 pig sera were collected and stored by the unit from pig farms, including 292 pig negative sera collected before foot-and-mouth disease vaccine immunization, 66 sera collected 28 days after immunization of bivalent inactivated vaccine of foot-and-mouth disease virus of pigs type O and type A (Re-O / MYA98 / JSCZ / 2013 strain + Re-A / WH / 09 strain), and 14 sera collected 28 days after immunization of bivalent synthetic peptide vaccine of foot-and-mouth disease virus of pigs type O and type A (polypeptide 2700+2800+MM13). The background of all sera was titrated according to the virus neutralization test required by the World Organization for Animal Health (OIE). The determination results of each kit are shown in Table 7. The solid-phase blocking ELISA kit for foot-and-mouth disease virus type A antibody was used to detect pig negative sera and sera of pigs immunized with bivalent inactivated vaccine of foot-and-mouth disease virus of pigs type O and type A, and 60 positive samples were detected, and 298 negative samples were detected, with a total coincidence rate of 95.5% with the kit. The liquid-phase blocking ELISA antibody detection kit for foot-and-mouth disease virus type A was used to detect pig negative sera and sera of pigs immunized with bivalent inactivated vaccine of foot-and-mouth disease virus of pigs type O and type A, and 64 positive samples were detected, and 294 negative samples were detected, with a total coincidence rate of 98.8% with the kit. For the sera of pigs immunized with bivalent synthetic peptide vaccine of foot-and-mouth disease virus of pigs type O and type A, the positive rates of the two commercialized kits were 42.9% and 57.1% respectively, but the antibody positive rate of the kit reached 92.9%, indicating that the overall immunization effect of the vaccine was good, and the kit using foot-and-mouth disease virus VP1 structural protein polypeptide could better reflect the antibody level of the vaccine.

[0148] Table 7 Kit clinical detection results

[0149]

[0150] Example 4, specificity analysis

[0151] At present, all the FMD A type vaccines in China are used with O type vaccine multivalent, including FMD OA bivalent synthetic peptide vaccine, FMD OA bivalent inactivated vaccine, and the structural protein amino acid sequences of FMD O type A type virus are relatively high, such as O / MYA98 / JSCZ / 2013 strain and A / WH / 09 strain VP1 amino acid sequence similarity reaches 73.83%, and the VP2-VP4 sequence similarity is more than 85%, which puts forward high requirements for the detection specificity of FMD antibody detection kit, and solving the specificity is one of the biggest difficulties of FMD virus VP1 structural protein polypeptide ELISA antibody detection kit development.

[0152] The 60 specific quality control sera were tested by using the kit and the commercial pig FMD A type virus VP1 structural protein ELISA diagnostic kit disclosed in the literature (Kit selection and evaluation for detection of antibody level after immunization of pig FMD virus synthetic peptide vaccine). The 60 specific quality control sera were prepared and stored by the unit, and were composed of FMD virus O type vaccine immune antibody serum and common pig disease antibody serum which were prone to cross reaction, including 20 FMD virus O type inactivated vaccine (Re-O / MYA98 / JSCZ / 2013 strain) immune antibody positive serum, 20 pig FMD O type synthetic peptide vaccine (polypeptide 2600+2700+2800) immune antibody positive serum, 10 pig FMD negative serum, 2 pig parvovirus inactivated vaccine immune antibody positive serum, 2 pig pest live vaccine immune antibody positive serum, 2 pig reproductive and respiratory syndrome virus live vaccine immune antibody positive serum, 2 pig pseudorabies virus live vaccine immune antibody positive serum, and 2 pig circovirus type 2 inactivated vaccine immune antibody positive serum. The detection results are shown in Table 8, and the detection of other common pig disease antibodies and pig negative serum by the two kits were all negative, indicating that the general specificity was good; but the detection of O type inactivated vaccine antibodies and O type synthetic peptide vaccine antibodies by the commercial kit detected 3 and 5 positive, respectively, indicating that there was a certain proportion of cross reaction; and the detection results of the above serum by the kit were all negative, indicating that the kit could effectively reduce the cross reaction of O type vaccine immune antibody, and was more conducive to the monitoring of the real immune effect of FMD A type vaccine in clinic.

[0153] Table 8 Kit specificity analysis

[0154]

[0155] The above describes the specific embodiments of the present application. It should be understood that the present application is not limited to the above specific embodiments, and those skilled in the art can make various modifications or modifications within the scope of the claims, which does not affect the essential content of the present application.

Claims

1. A foot-and-mouth disease virus type A epitope polypeptide, the structure of which is as follows: a peptide with the amino acid sequence VYNGTSKYSAPATRRGDLGSLAARLAAQLPASGSS is simultaneously linked to the N-terminal amino group and the side chain amino group of Lys to form a binary branched peptide (VYNGTSKYSAPATRRGDLGSLAARLAAQLPASGSS)2K.

2. A foot-and-mouth disease virus type A epitope polypeptide, the structure of which is as follows: A peptide with the amino acid sequence VYSGTSKYSAPQNRRGDSGPLAARLAAQLPASGSS is simultaneously linked to both the N-terminal amino group and the side chain amino group of Lys to form a binary branched peptide (VYSGTSKYSAPQNRRGDSGPLAARLAAQLPASGSS)2K.

3. A foot-and-mouth disease virus type A epitope polypeptide, the structure of which is as follows: A peptide with the amino acid sequence VYNGTTKYSTGNAGRRGDLGSLAARVAAQLPASGSS is simultaneously linked to both the N-terminal amino group and the side chain amino group of Lys to form a binary branched peptide (VYNGTTKYSTGNAGRRGDLGSLAARVAAQLPASGSS)2K.

4. An antigen-coated plate, the coated plate containing three epitope polypeptides 1-3, wherein epitope polypeptide 1 is the epitope polypeptide of claim 1, epitope polypeptide 2 is the epitope polypeptide of claim 2, and epitope polypeptide 3 is the epitope polypeptide of claim 3.

5. A method for preparing an antigen-coated plate according to claim 4, characterized in that, The method includes the following steps: S1. Epitope polypeptides 1-3 were synthesized using solid-phase synthesis methods respectively; S2. Mix the three epitope peptides 1-3, dilute them, add them to the wells of an enzyme-linked reaction plate, and then coat, block, and dry them to obtain the antigen-coated plate.

6. The method for preparing the antigen-coated plate according to claim 5, characterized in that, In step S2, the mass ratio of epitope polypeptide 1, epitope polypeptide 2, and epitope polypeptide 3 is 1:1:1 to 1:3:3, or 1:1:1 to 3:1:3, or 1:1:1 to 3:3:

1.

7. The method for preparing the antigen-coated plate according to claim 5, characterized in that, In step S2, the coating solution is a 0.1 mol / L carbonate-bicarbonate buffer solution with a pH of 9.55–9.65, wherein the carbonate-bicarbonate buffer solution contains 0.02%–0.08% DMSO and 0.3%–1% Tween 20.

8. A foot-and-mouth disease virus type A ELISA antibody detection kit, the kit comprising the antigen-coated plate as described in claim 4.

9. The foot-and-mouth disease virus type A ELISA antibody detection kit according to claim 8, characterized in that, The kit also includes an enzyme conjugate, an enzyme conjugate diluent, a sample diluent, a colorimetric solution, a stop solution, and a 25x wash solution.

10. The use of the antigen-coated plate as described in claim 4 in the preparation of a foot-and-mouth disease virus type A ELISA antibody detection kit, characterized in that, The kit is used to detect the level of foot-and-mouth disease type A antibodies.

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