阅读说明：本技术 一种狐狸逆转录病毒毒株及其应用 (Fox retrovirus strain and application thereof ) 是由 王玉茂 于 2021-08-23 设计创作，主要内容包括：本发明提供了一种狐狸逆转录病毒毒株及其应用,该毒株的保藏编号为CGMCC No.21898,分类命名为γ逆转录病毒,该狐狸逆转录病毒毒株于2021年06月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址：北京市朝阳区北辰西路1号院,邮编：100101；将狐狸逆转录病毒毒株进行应用,具体为制成药物后应用和/或诊断制剂中的应用。通过将该毒株进行应用,使对由狐狸逆转录病毒引起的疾病的预防、治疗等更具目标性,提高检测效率、预防效果和治疗效果。(The invention provides a fox retrovirus strain and application thereof, the preservation number of the strain is CGMCC No.21898, the strain is classified and named as gamma retrovirus, the fox retrovirus strain is preserved in the China general microbiological culture Collection center at 23.06.2021, and the address is as follows: west road No.1, north-west, morning-sun, beijing: 100101; the fox retrovirus strain is applied, in particular to the application after being prepared into a medicament and/or the application in a diagnostic preparation. By applying the strain, the prevention, treatment and the like of diseases caused by the fox retrovirus are more targeted, and the detection efficiency, the prevention effect and the treatment effect are improved.)

1. A fox retrovirus strain is characterized in that the fox retrovirus strain is gamma retrovirus PreXMRV-20 with the preservation number of CGMCC No.21898 and is classified and named as gamma retrovirus; the fox retrovirus strain is preserved in China general microbiological culture Collection center at 23.06.23.2021, and the address is as follows: west road No.1, north-west, morning-sun, beijing: 100101.

2. the fox retrovirus strain according to claim 1, wherein the gene sequence of the gamma retrovirus PreXMRV-20 comprises the gene segments shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.

3. Use of a fox retrovirus strain according to claim 1 or 2 for the preparation of a medicament.

4. Use according to claim 3, wherein the medicament is a medicament for the prophylaxis and/or treatment of a fox retroviral disease.

5. The use of claim 3, wherein the medicament is a vaccine, hyperimmune serum preparation, hyperimmune plasma preparation and/or antibody preparation made from attenuated or inactivated strains.

6. Use of a fox retrovirus strain according to claim 1 or 2 for the preparation of a diagnostic formulation for a fox retroviral disease.

7. The use of claim 6, wherein the diagnostic formulation comprises an antigen detection kit and an antibody detection kit.

8. The use of claim 7, wherein the antigen in the antigen detection kit is selected from the group consisting of a viral strain, a lytic component of a viral strain, a genetically engineered protein of a viral strain, or a polypeptide of a viral strain; the antibody in the antibody detection kit is selected from a virus strain, a lysis component of the virus strain, a genetically engineered protein of the virus strain or a monoclonal antibody or a polyclonal antibody prepared from a polypeptide of the virus strain.

Technical Field

The invention relates to the technical field of viruses, in particular to a fox retrovirus strain and application thereof.

Background

The disease caused by the fox retrovirus is a disease with the infection rate of a population reaching 60 percent, the infection rate in an attack group is about 5 to 10 percent, the kidney is swollen, the fox after the disease is developed grows slowly and becomes thin, the hair color becomes light and rough, and the produced skin has no utilization value due to small area and poor quality; at present, the fox retrovirus is less recognized, so that the prevention and treatment of the disease have certain difficulty.

The method is a scientific and reasonable method for determining diseases by gene sequencing and gene comparison through instrument equipment, and specifically comprises the steps of extracting a gene sample, analyzing the gene sequence of the sample to obtain a gene fragment, recognizing viruses and knowing the viruses through the gene fragment, and further preventing and controlling the viruses.

In order to enhance the cognition of the fox retrovirus and improve the prevention capability of the fox retrovirus to the disease, the important significance is realized by carrying out gene extraction and gene comparison on the retrovirus so as to realize the virus from a gene layer.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a fox retrovirus strain and application thereof, wherein the preservation number of the strain is CGMCC No.21898, and the gene sequence of the strain comprises gene segments shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the fox retrovirus strain is applied, in particular to the application after being prepared into a medicament and/or the application in a diagnostic preparation. By applying the strain, the prevention, treatment and the like of diseases caused by the fox retrovirus are more targeted, and the detection efficiency, the prevention effect and the treatment effect are improved. By applying the strain, the prevention and control of diseases generated by the virus can be improved, and the treatment target can be improved.

The technical scheme of the invention is as follows:

a fox retrovirus strain, which is gamma retrovirus PreXMRV-20 with the preservation number of CGMCC No.21898 and is classified and named as gamma retrovirus; the fox retrovirus strain is preserved in China general microbiological culture Collection center at 23.06.23.2021, and the address is as follows: west road No.1, north-west, morning-sun, beijing: 100101.

further, the gene sequence of the strain comprises gene segments shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, which are respectively as follows:

SEQ ID No.1：

TATGGAGTCATGTCGATGAGGAATGATTATATCTGGTTGTCGATCTTTGGCCACGGGGTCAAAGGGGGTGTATATATGGTAGCCTTCCATTAATTTTTCCAGAAAACCTGCCGGCGTCTCATCTTTCCCCTGCACTATAGCACGTACCTTTGCCAAATTGGTGGGGCGTCGCCCTGCCCCTTTAAAACCGCTAGGAGTACCTGGGGATAGAGACGGAGCCGCTCCCTACCAGCAGCAGTGTCGTAGTCCCAGGTCGGGCGAACCAAGGGGAAAACATCCTCAATTTCATTAGGCAACTGAGTGGA；

SEQ ID No.2：

CCGAGTCATGTCGATGAGGAATGATTACATCTGGTTGTCGATCTTTGGCCACGGGGTCAAAGGGGGTGTATATATGGTAGCCTTCCATTAATTTTTCCAGAAAACCTGCCGGCGTCTCATCTTTCCCCTGCACTATAGCACGTACCTTTGCCAAATTGGTGGGGCGTCGCCCTGCCCCTTTAAAACCGCTAGGAGTACCTGGGGATAGAGACGGAGCCGCTCCCTACCAGCAGCAGTGTCGTAGTCCCAGGTCGGGCGAACCAAGGGGAAAACATCCTCAATTTCATTAGGAAACTGAGTGGA；

SEQ ID No.3：

GGGTTCGGGTGGTCCCGACCTGGGACTACGACACTGCATGCTGGTAGGGAGCGGCTCCGTCTCTATCCCCAGGTACTCCTAGCGGGTCTCAAGGGGCAGGGCGATGCCCCACCAATTTGGCAAAGGTACGTGCTATAGTGCAGGGGAAAGATGAGACGCCAGCAGGATTTCTGGAAAAATTAATGGAAGGCTACCATATGTACACCCCCTTTGACCCCTTGGCCAAAGATCGGCAACCAGATGTAATCATGTCCTTCATCGGACAATAGGCCTCGGGTATTTGTAATAAGTTAGAGCGGTTAGAAGA；

the strain was isolated from a renal tissue sample of a diseased fox.

The fox retrovirus strain is applied to the preparation of medicines.

Preferably, the medicament is particularly a medicament for preventing and/or treating fox retroviral diseases.

Preferably, the medicament is a vaccine, a hyperimmune serum preparation, a hyperimmune plasma preparation and/or an antibody preparation prepared by attenuating or inactivating the strain.

The fox retrovirus diseases are fox diseases caused by fox retrovirus, such as slow growth, reproduction disorder, organ swelling, especially kidney swelling, which seriously affect fox breeding.

The fox retrovirus strain is applied in preparing fox retrovirus disease diagnosis preparation.

Preferably, the diagnostic formulation comprises an antigen detection kit and an antibody detection kit.

Preferably, the antigen in the antigen detection kit is selected from a virus strain, a cleavage component of a virus strain, a genetically engineered protein of a virus strain, or a polypeptide of a virus strain; the antibody in the antibody detection kit is selected from a virus strain, a lysis component of the virus strain, a genetically engineered protein of the virus strain or a monoclonal antibody or a polyclonal antibody prepared from a polypeptide of the virus strain.

Compared with the prior art, the invention has the beneficial effects that:

1. in the invention, three gene segments (SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3) of the gene sequence of the fox retrovirus strain are determined by performing gene extraction and sequencing on the kidney tissue of the diseased fox, and the preservation number of the fox retrovirus strain is CGMCC No. 21898.

2. The genome sequencing of the fox retrovirus is carried out, so that the cognition degree of the fox retrovirus is improved, a direction is provided for the prevention and research of fox retrovirus diseases from a gene level, and the blindness of the prevention and treatment of the fox retrovirus is reduced.

3. The strain provided by the invention can effectively prevent diseases caused by fox retrovirus by preparing the strain into a vaccine medicament, and can effectively treat the diseases caused by the fox retrovirus by preparing the strain into a hyperimmune serum preparation, a hyperimmune plasma preparation and/or an antibody preparation.

4. The strain provided by the invention can improve the timeliness, effectiveness and accuracy of fox retrovirus detection and improve the prevention and treatment of fox retrovirus diseases by being prepared into a detection preparation.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a phylogenetic tree constructed by comparing the virus Gene sequence obtained by the present invention with Gene Bank;

FIG. 2 is a phylogenetic tree constructed by comparing the virus gene sequence obtained by the present invention with the reported gamma retrovirus;

FIG. 3 shows the morphology of the viral particles obtained in example 3 under an electron microscope.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the embodiment of the invention, the fox retrovirus tissue sample is taken from a diseased fox in Shandong Weifang, and the preparation method of the tissue sample is as follows:

(1) taking diseased fox tissues, grinding by using a grinder, freeze thawing for three times, centrifuging for 5min at 10000rpm, discarding the precipitate, and taking the supernatant to obtain a sample I;

(2) mixing the sample I with the extracting solution, extracting and separating to obtain a sample II;

the composition of the extract was as follows:

200mM Tris-HCl (pH 8.0), 500mM NaCl, 5mM EDTA, 1% sodium dodecyl sulfate (SDS, W/V), 2% lithium dodecyl sulfate (LLS, W/V) and 1% betaine (W/V);

(3) processing the protein in the sample II to obtain a tissue sample;

wherein the tissue sample is selected from brain tissue, kidney tissue, liver tissue, lung tissue or spleen tissue of diseased fox;

the diseased fox has the following characteristics: hydrocephalus, enlarged kidney, slight bleeding spot in liver, secretion in eye and slight swelling of lung surface.

Example 1

1. Obtaining fox retrovirus:

the separation and purification method of the fox retrovirus comprises the following steps:

(1) preparing a renal tissue sample according to a preparation method of a fox retrovirus tissue sample by taking a renal tissue of a diseased fox;

(2) performing genome extraction on the kidney tissue sample obtained in the step (1), and collecting an extraction product; the genome extraction process is carried out according to the instruction of a virus genome Dna/Rna extraction kit (Thermo Fisher Scien tific); detecting the extracted product by 1% agarose gel electrophoresis, and storing at-20 deg.C for use;

(3) taking the extracted product in the step (2), sending the extracted product to Shijiazhuang Boridii biotechnology limited for genome sequencing, and specifically, carrying out Paired-End sequencing on DNA of a sample by adopting Illumina sequencing technology; the obtained sequence fragments are assembled by MEGAHIT through sequence splicing, virus sequences are screened from the assembly result, and the virus database result is compared through genome sequencing, so that the homology of the virus and a PreXMRV-1 provirus strain (GenBank accession number NC-007815.2) reaches 82.587 percent, and the virus is named as a PreXMRV-20 strain. After sequencing, the gene sequence of the obtained fox retrovirus comprises three gene segments which are respectively as follows:

SEQ ID No.1：

TATGGAGTCATGTCGATGAGGAATGATTATATCTGGTTGTCGATCTTTGGCCACGGGGTCAAAGGGGGTGTATATATGGTAGCCTTCCATTAATTTTTCCAGAAAACCTGCCGGCGTCTCATCTTTCCCCTGCACTATAGCACGTACCTTTGCCAAATTGGTGGGGCGTCGCCCTGCCCCTTTAAAACCGCTAGGAGTACCTGGGGATAGAGACGGAGCCGCTCCCTACCAGCAGCAGTGTCGTAGTCCCAGGTCGGGCGAACCAAGGGGAAAACATCCTCAATTTCATTAGGCAACTGAGTGGA；

SEQ ID No.2：

CCGAGTCATGTCGATGAGGAATGATTACATCTGGTTGTCGATCTTTGGCCACGGGGTCAAAGGGGGTGTATATATGGTAGCCTTCCATTAATTTTTCCAGAAAACCTGCCGGCGTCTCATCTTTCCCCTGCACTATAGCACGTACCTTTGCCAAATTGGTGGGGCGTCGCCCTGCCCCTTTAAAACCGCTAGGAGTACCTGGGGATAGAGACGGAGCCGCTCCCTACCAGCAGCAGTGTCGTAGTCCCAGGTCGGGCGAACCAAGGGGAAAACATCCTCAATTTCATTAGGAAACTGAGTGGA；

SEQ ID No.3：

GGGTTCGGGTGGTCCCGACCTGGGACTACGACACTGCATGCTGGTAGGGAGCGGCTCCGTCTCTATCCCCAGGTACTCCTAGCGGGTCTCAAGGGGCAGGGCGATGCCCCACCAATTTGGCAAAGGTACGTGCTATAGTGCAGGGGAAAGATGAGACGCCAGCAGGATTTCTGGAAAAATTAATGGAAGGCTACCATATGTACACCCCCTTTGACCCCTTGGCCAAAGATCGGCAACCAGATGTAATCATGTCCTTCATCGGACAATAGGCCTCGGGTATTTGTAATAAGTTAGAGCGGTTAGAAGA；

the fox retrovirus strain is named as PreXMRV-20, and is preserved with the preservation information as follows: the preservation number of the strain is CGMCC No.21898, and the strain is classified and named as gamma retrovirus; the fox retrovirus strain is preserved in China general microbiological culture Collection center at 23.06.23.2021, and the address is as follows: west road No.1, north-west, morning-sun, beijing: 100101.

and respectively preparing a brain tissue sample, a liver tissue sample, a lung tissue sample and a spleen tissue sample of the diseased fox, and carrying out RT-PCR detection on the four different tissue samples and the kidney tissue sample together, wherein the detection results show that the brain tissue sample, the liver tissue sample, the lung tissue sample and the spleen tissue sample of the diseased fox are consistent with the detection results of the kidney tissue sample, so that the diseased fox also contains the fox retrovirus PreXMRV-20 in the brain tissue, the liver tissue, the lung tissue and the spleen tissue.

Example 2

The Gene fragment obtained by metagenome sequencing is processed by MEGA7 software, and is subjected to Gene homology comparison and genetic evolution analysis with the Gene sequence published on the Gene Bank. The virus gene is sequenced and sequence alignment is carried out, and a genetic evolutionary tree is constructed, which is shown in figure 1. The evolutionary tree shows that the virus has the highest homology with PreXMRV-1 and also has higher homology with PreXMRV-2 and MLV. This suggests that the virus may be a heterologous strain of MLV together with XMRV. The genetic evolutionary tree constructed by the reported gamma retrovirus is shown in figure 2, and the virus has the highest homology with bat-derived RfRV by combining figure 2, which indicates that the virus is probably derived from bat and the result is to be verified by further experiments.

Example 3

Taking the grinding fluid of the kidney and lung tissues of the diseased fox, centrifuging at 8000rpm for 10min to remove tissue fragments, taking the supernatant, adding 0.5% phosphotungstic acid solution, and observing the morphological characteristics by negative staining with an electron microscope, wherein the figure is shown in figure 3, the A figure in figure 3 is a particle morphology diagram of the virus in the kidney tissues under the electron microscope, and the B figure is a particle morphology diagram of the virus in the lung tissues under the electron microscope. The results show that the virus in the tissues of kidney (panel A) and lung (panel B) is in a non-capsular, polygonal structure with a diameter of about 90nm, and the virus-like particles are consistent with the morphology and size of XMRV.

Although the present invention has been described in detail by referring to the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

SEQUENCE LISTING

<110> Shandong province Binzhou animal husbandry veterinary research institute

<120> fox retrovirus strain and application thereof

<130> 2021

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 305

<212> DNA

<213> Gamma retrovirus

<400> 1

tatggagtca tgtcgatgag gaatgattat atctggttgt cgatctttgg ccacggggtc 60

aaagggggtg tatatatggt agccttccat taatttttcc agaaaacctg ccggcgtctc 120

atctttcccc tgcactatag cacgtacctt tgccaaattg gtggggcgtc gccctgcccc 180

tttaaaaccg ctaggagtac ctggggatag agacggagcc gctccctacc agcagcagtg 240

tcgtagtccc aggtcgggcg aaccaagggg aaaacatcct caatttcatt aggcaactga 300

gtgga 305

<210> 2

<211> 303

<212> DNA

<213> Gamma retrovirus

<400> 2

ccgagtcatg tcgatgagga atgattacat ctggttgtcg atctttggcc acggggtcaa 60

agggggtgta tatatggtag ccttccatta atttttccag aaaacctgcc ggcgtctcat 120

ctttcccctg cactatagca cgtacctttg ccaaattggt ggggcgtcgc cctgcccctt 180

taaaaccgct aggagtacct ggggatagag acggagccgc tccctaccag cagcagtgtc 240

gtagtcccag gtcgggcgaa ccaaggggaa aacatcctca atttcattag gaaactgagt 300

gga 303

<210> 3

<211> 307

<212> DNA

<213> Gamma retrovirus

<400> 3

gggttcgggt ggtcccgacc tgggactacg acactgcatg ctggtaggga gcggctccgt 60

ctctatcccc aggtactcct agcgggtctc aaggggcagg gcgatgcccc accaatttgg 120

caaaggtacg tgctatagtg caggggaaag atgagacgcc agcaggattt ctggaaaaat 180

taatggaagg ctaccatatg tacaccccct ttgacccctt ggccaaagat cggcaaccag 240

atgtaatcat gtccttcatc ggacaatagg cctcgggtat ttgtaataag ttagagcggt 300

tagaaga 307