阅读说明：本技术 一种抗h9n2亚型禽流感病毒的多肽及其应用 (Polypeptide for resisting H9N2 subtype avian influenza virus and application thereof ) 是由 盛晓丹 黄迪海 秦卓明 徐怀英 郭卉 秦春芝 于 2021-07-08 设计创作，主要内容包括：本发明属于动物医学分子免疫学技术领域,尤其涉及一种抗H9N2亚型禽流感病毒的多肽及其应用。通过抗H9N2亚型禽流感病毒的多肽,其氨基酸序列：RRKKWLVFFVIFYFFR,应用于制备治疗禽流感病毒的药物中,也可与抗病毒中药,干扰素,流感病毒抗体,转移因子等的一种或几种进行组方,制备抗H9N2亚型禽流感病毒的产品。(The invention belongs to the technical field of animal medical molecular immunology, and particularly relates to a polypeptide for resisting H9N2 subtype avian influenza virus and application thereof. By the polypeptide of anti-H9N 2 subtype avian influenza virus, the amino acid sequence: RRKKWLVFFVIFYFFR, it can be used for preparing medicine for treating avian influenza virus, and can be formulated with one or more of antiviral Chinese medicinal materials, interferon, influenza virus antibody, transfer factor, etc. to prepare product for resisting H9N2 subtype avian influenza virus.)

1. A polypeptide for resisting H9N2 subtype avian influenza virus is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO 1; amino acid sequence: RRKKWLVFFVIFYFFR are provided.

2. A nucleotide sequence encoding the epitope polypeptide of claim 1; the nucleotide sequence is shown as SEQ ID NO. 2; the nucleotide sequence is as follows: GTCGTAAAAAATGGCTGGTTTTTTTTGTTATTTTTTATTTTTTTCGT are provided.

3. A recombinant expression vector carrying the nucleotide of claim 2.

4. Use of the polypeptide of claim 1 for the manufacture of a medicament for the treatment of avian influenza virus.

5. The use according to claim 4, wherein the avian influenza virus is chicken avian influenza virus.

Technical Field

The invention belongs to the technical field of animal medical molecular immunology, and particularly relates to a polypeptide for resisting H9N2 subtype avian influenza virus and application thereof.

Background

Avian Influenza (AI) is a collective term for Avian infectious diseases caused by Influenza a viruses that occur in various poultry and wild birds. They are classified into many subtypes according to the antigenicity of Hemagglutinin (HA) and Neuraminidase (NA) on the surface of influenza A virus particles. According to the pathogenicity of the clinical influenza A virus, the avian influenza can be divided into three types, namely high pathogenicity, low pathogenicity and non-pathogenicity. Although the H9N2 subtype avian influenza belongs to low-pathogenicity avian influenza, the avian influenza seriously harms the production of the chicken industry, is an important reason for high death and culling rate of commercial broilers, and not only causes reduction of immunity of commercial broilers, respiratory symptoms and secondary escherichia coli, but also can cause serious egg laying reduction and egg quality reduction of breeding hens and laying hens.

The domestic prevention and control of H9N2 avian influenza mainly depends on vaccines, but because the virus mutation speed is high, the virus strain needs to be replaced every 2-3 years. The traditional Chinese medicine is used for treating epidemic diseases, but the traditional Chinese medicine has generally weak antiviral activity, is mainly used for comprehensively conditioning through the functions of clearing away heat and toxic materials, regulating immunity and the like, has no approval for veterinary antiviral chemicals and biological products, and is lack of effective prevention and treatment medicines.

Tkip is a mimic of cytokine signal inhibitory factor (SOCS) protein, is a tyrosine kinase inhibitor consisting of 12 amino acids (WLVFFVIFYFFR), and has anti-inflammatory activity. In 2009, Ahmed, c.m. et al reported that Tkip has anti-poxvirus activity in vitro and in vivo in mice. In 2012, Marlynne Q. and the like designed and synthesized Tkip series derived peptides FP 2-FP 9 in order to change the water solubility, half-life and stability of Tkip (FP 1), and have certain inhibitory effect on influenza virus strains of subtypes such as A/WSN/33(H1N1), PR8/Eng09(H1N1), PR8/Vic (H3N2), PR8/Viet (H5N1) and the like. Wherein, the IC of FP4 polypeptide (amino acid sequence: RRKKWLVFFVIFYFFR) to the above 4 viruses₅₀The antiviral effect is better at the cellular level when the antiviral effect is respectively 0.00004 mu M, 0.0778 mu M, 0.0535 mu M and 0.1325 mu M. However, no report on influenza virus subtype H9N2 has been made.

Disclosure of Invention

Aiming at the serious situation of H9N2 subtype avian influenza epidemic situation in China at the present stage and the problem that no effective prevention and treatment medicine exists, the invention prepares the product for resisting H9N2 subtype avian influenza virus by the polypeptide for resisting H9N2 subtype avian influenza virus, which can be applied to the preparation of the medicine for treating avian influenza virus and can also be combined with one or more of antiviral traditional Chinese medicines, interferon, influenza virus antibodies, transfer factors and the like.

The technical scheme of the invention is as follows:

a polypeptide for resisting H9N2 subtype avian influenza virus, the amino acid sequence of the polypeptide is shown in SEQ ID NO 1; amino acid sequence: RRKKWLVFFVIFYFFR are provided.

A nucleotide encodes the epitope polypeptide; the nucleotide sequence is SEQ ID NO. 2; the nucleotide sequence is as follows: CGTCGTAAAAAATGGCTGGTTTTTTTTGTTATTTTTTATTTTTTTCGT are provided.

A recombinant expression vector carries the nucleotide.

The application of the polypeptide in preparing the medicine for treating the avian influenza virus.

Preferably, the avian influenza virus is chicken avian influenza virus.

The invention has the advantages of

1. The chicken embryo test of H9N2 AIV resistance is carried out, and the survival rate of the chicken embryo can reach 100 percent at most after FP4 polypeptide is mixed with H9N2 AIV and is respectively inoculated with 0.001-0.016 mg of FP4 polypeptide and 200EID 50 virus according to each chicken embryo.

2. Has great application prospect in preparing the medicine for treating the avian influenza virus, and can be used for preparing the product for resisting the H9N2 subtype avian influenza virus by composing with one or more of antiviral traditional Chinese medicines, interferon, influenza virus antibody, transfer factor and the like.

Detailed Description

Example 1

A polypeptide for resisting H9N2 subtype avian influenza virus, the amino acid sequence of the polypeptide is shown in SEQ ID NO 1; amino acid sequence: RRKKWLVFFVIFYFFR are provided.

A nucleotide encodes the epitope polypeptide; the nucleotide sequence is SEQ ID NO. 2; the nucleotide sequence is as follows: CGTCGTAAAAAATGGCTGGTTTTTTTTGTTATTTTTTATTTTTTTCGT are provided.

A recombinant expression vector carries the nucleotide.

EXAMPLES Effect example 1

Application in treating avian influenza

The implementation scheme is as follows:

1 materials and methods

1.1 main experimental materials and H9N2 subtype avian influenza virus isolate (number: 402, H9N2 AIV for short) of experimental animals are preserved in the laboratory; a 10-day-old SPF chick embryo provided by Shandong Haotai laboratory animal Breeding Co., Ltd; FP4 polypeptide (amino acid sequence: RRKKWLVFFVIFYFFR) was synthesized by the Wahuada gene.

The main reagent is a streptomycin double-resistant solution (raw product).

Recovering virus, thawing diluted H9N2 AIV isolate, inoculating to 10-day-old chick embryo, standing at 37 deg.C for 3 days, collecting allantoic fluid, diluting with physiological saline to 200EID₅₀And (5) standby.

Preparation of test drugs

1.4.1 preliminary experiments FP4 polypeptide 1mg was weighed out accurately, dissolved in 50. mu.L ethanol, diluted to 1mg/mL stock solution with 950. mu.L sterile water and split charged. Mixing 200 μ L stock solution with 300 μ L sterile water and 500 μ L double antibody solution, preparing 0.2mg/mL FP4 solution as working solution, and standing at 37 deg.C for 1 h.

1.4.2 formal experiment weighing 5mg FP4 polypeptide, adding 50 μ L ethanol to dissolve polypeptide, adding 950 μ L sterile water and 1mL double antibody to dilute to 2.5mg/mL stock solution, and packaging.

Toxicity attacking protection experiment

1.5.1 grouping and handling of test chick embryos are shown in tables 1 and 2.

TABLE 1 preliminary Experimental protocol

Table 2 official experimental protocol

1.5.2 determination of death and Hemagglutination (HA) Titers

Standing and culturing chicken embryos at 37 ℃ for 120 days, observing death conditions every day, discarding the chicken embryos dying within 24 hours, inoculating viruses for 120 hours, collecting allantoic fluid of each group of chicken embryos, respectively measuring HA titer of each group, and recording and counting results.

Results

2.1 preliminary experiments

Within 48h, the chick embryos of the positive control group and the chick embryos of the 2 groups die earliest, within 72h, the chick embryos of the 1 group and the chick embryos of the 2 groups and the positive control group die in 96h, and the survival rate of the 3 group and the negative control group is 100% at 120 h. The results of virus Hemagglutination (HA) titers (Table 3) showed that the titers of the mixed vaccinated groups were all 0, with the best results, and that the high dose vaccinated group first, followed by vaccinated group, was not significantly different from the control.

TABLE 3 chick embryo H9N2 HA potency and survival Rate in the Pre-Experimental group

2.2 official experiment

Within 48h, chick embryos of 1 group, 2 groups and a positive control group die at the earliest, chick embryos of 3 groups within 72h die, chick embryos of 1 group, 2 group, 3 group and the positive control group die at 96h, and survival rates of 5 and 6 mixed groups and the negative control group are 100% at 120 h. The results of virus Hemagglutination (HA) titers showed (table 4), with the titers of both the medium and high dose mixed vaccination groups being 0, with the best results, followed by 1.3 for the low dose mixed group; the high dose prevention (3 groups) has the titer reduced by 1.1, the death time is delayed, a certain antiviral effect is achieved, and the low and medium dose prevention groups have no obvious antiviral effect.

TABLE 4 chick embryo H9N2 HA potency and survival Rate in the Pre-Experimental group

3 conclusion

3.1 safety FP4 polypeptide is safe to chick embryos.

The effective FP4 polypeptide HAs the effect of inhibiting the virus from infecting chick embryos, the most effective mode is that the effective dose is more than or equal to 0.001 mg/embryo, the survival rate of the chick embryos reaches 87.5% -100%, and the action mechanism of the effective FP4 polypeptide is probably that the FP4 polypeptide is targeted and combined with the HA head (HA 1) of H9N2 AIV, so that the virus loses the capacity of combining with host cell receptors. The inoculation of FP4 alone at 0.016 mg/embryo followed by H9N2 AIV did not improve survival, but overall reduced antibody titers and was less effective against viruses. After H9N2 AIV inoculation, FP4 polypeptide of 0.016 mg/embryo is inoculated independently, and no obvious antiviral effect is achieved.

The FP4 polypeptide has obvious antiviral effect, but the polypeptide is easy to be degraded by enzyme in animal body after oral administration, so the drug can be used in poultry industry by intramuscular injection, subcutaneous injection, nasal drip and other modes, and can be used with antiviral traditional Chinese medicine, interferon, influenza virus antibody, transfer factor and other modes to prepare the anti-H9N 2 AIV veterinary drug.

EXAMPLES Effect example 2

Application of polypeptide for resisting H9N2 subtype avian influenza virus in preventing H9N2 avian influenza

1 materials and methods

1.1 main experimental materials and H9N2 subtype avian influenza virus isolate (No. 402, H9N2 AIV, EID)₅₀Is 10^7.6250.2 ml) was kept in the laboratory; 1 day old SPF chicks, provided by the eastern Shandong poultry institute; FP4 polypeptide (amino acid sequence: RRKKWLVFFVIFYFFR) was synthesized by the Wahuada gene.

Preparation of test drugs

Weighing 6mg FP4 polypeptide, adding 100 μ L ethanol to dissolve polypeptide, adding appropriate amount of sterile water to dilute to 3mg/mL stock solution, subpackaging, and freezing for storage. 0.1ml stock solution is taken before use and diluted to 0.03mg/ml to be used as working solution for standby.

Grouping of test chickens

40 chicks were randomly divided into 5 groups of 8 chickens, each group was a FP4 low dose group, a FP4 medium dose group, a FP4 high dose group, a positive control group, and a negative control group, and the specific grouping and treatment were shown in table 5.

TABLE 5 grouping and handling of laboratory chickens

1.4 Collection and treatment of pathological Material

The test lasts for 14 days, the clinical performance of the chickens is observed daily, cloacal mucus is collected by cotton swabs for each experimental group daily, viral RNA in the mucus is extracted, and RT-PCR is carried out to identify H9N2 pathogen in the mucus.

2 results

2.1 clinical symptoms of test chickens

After the positive control chicken is inoculated with H9N 248H, the results show that the feed intake is reduced, the spirit is depressed, the chicken is dull and hair-fried, secretions flow out of nasal cavities of a few chickens, and no chicken dies in the whole grouping experiment. Few chickens in the low-dose group had lacrimation, necking and standing symptoms, and the medium-dose and high-dose groups had no obvious symptoms.

2.2 RT-PCR results

After the mucus RNA samples treated by the method 1.4 are identified by RT-PCR, virus RNA is detected only in the 1 st day after virus attack of medium and high dose groups, virus RNA is detected only in the 1, 2 and 3 days after virus attack of low dose groups, virus RNA is detected in the 1 st to 7 th days after virus attack of positive control groups, and no virus is detected in the negative control groups, and the specific results are shown in Table 6.

TABLE 6 results of detecting cloacal swabs by RT-PCR

3 conclusion

The chicken is pretreated by FP4 solution in an eye-dropping and nose-dropping manner before being infected with H9N2 virus, and the virus RNA of the cloacal swab is detected by an RT-PCR method, so that the FP4 can effectively inhibit the H9N2 virus from entering the chicken body through the respiratory mucosa, and a basis is provided for clinically preventing H9N2 subtype low-pathogenicity avian influenza.

Sequence listing

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