CN116240295A - Molecular marker for chicken genetic resistance to B subgroup avian leukosis virus infection, screening/identifying method and application thereof - Google Patents

Abstract

The invention discloses a molecular marker for chicken genetic resistance to B subgroup avian leukosis virus infection, a screening/identifying method and application thereof, wherein the method comprises the following steps: extracting genome DNA of a sample to be detected, amplifying tvb receptor gene fragments, sequencing, and judging whether the 3747 th position of the tvb receptor gene has deletion of a base G. The method can rapidly and accurately judge whether the detection sample is an ALV-B resistant chicken or a susceptible chicken, can screen breeding materials for breeding an ALV-B infection resistant chicken variety/strain from chicken breeds (including local chicken breeds and commercial broiler strains) in China, and provides technical support for breeding a genetic ALV-B infection resistant chicken variety/strain.

Description

Molecular marker for chicken genetic resistance to B subgroup avian leukosis virus infection, screening/identifying method and application thereof

Technical Field

The invention relates to the technical field of poultry disease-resistant breeding, in particular to a molecular marker for chicken genetic resistance to B subgroup avian leukosis virus infection, a screening/identifying method and application thereof.

Background

Avian leukemia is a type of avian immunosuppressive neoplastic infectious disease caused by avian leukemia virus (Avian Leukosis Virus, ALV). Subgroup B avian leukosis virus (ALV-B) is the main pathogen causing avian leukosis of chicken flocks in China. ALV-B infection can cause the infected chicken group to die due to the occurrence of characteristic tumors, so that the production performance of the infected chicken group is reduced, the infected chicken group can be caused to generate serious immunosuppression, other viral diseases such as avian influenza, newcastle disease and the like and bacterial diseases such as escherichia coli and salmonella are extremely easy to cause huge economic losses for poultry industry in China. ALV-B is mainly transmitted vertically, the infection can be amplified from great ancestor generation (pure line), ancestor generation, parent generation and commodity generation by generation, the infection rate of each generation is increased by about 5% -20%,1 ancestor (pure line) chicken can be infected with ALV-B and then can be transmitted to 24 ten thousand commodity chickens, and the poultry leukemia has become the disease which is the greatest hazard to the poultry industry safety in China. At present, no commercial vaccine and no effective treatment method are available for preventing and controlling the disease, but the population is long in purification period (at least 8-10 years) and high in cost (about 500 yuan per chicken), and the purified negative chicken flock still faces the risk of re-infection of ALV, so that the purification effect of avian leukosis is difficult to maintain, and the disease is trapped in continuous fight of pathogen-animal-detection personnel. In recent years, epidemiological investigation and research and development of avian leukosis find that ALV-B occurs and is popular in different types of chickens such as local chickens, yellow feather chickens, white feather chickens, laying hens and the like in China, and the disease becomes one of serious diseases which threatens sustainable and healthy development of chicken raising industry (especially breeding hens industry) in China. Therefore, developing technical innovation for controlling avian leukosis and developing new strategies more suitable for preventing and controlling avian leukosis in China are urgent. Research has been carried out abroad to prove that research on disease-resistant breeding of avian leukosis from the aspect of host genetic resistance has become an effective strategy for preventing and controlling the disease.

Chicken Tvb receptor gene encodes a Tvb receptor protein, which mediates the invasion of ALV-B into host cells, followed by infection. Tvb receptor gene determines the susceptibility or resistance of host cells to ALV-B infection, and genetic mutations may result in complete deletion of expression of the Tvb receptor protein or expression of a defective Tvb receptor protein that is unsuitable as an ALV-B receptor, thereby causing infection of chickens against ALV-B. Therefore, the development and identification of the molecular markers for resisting ALV-B infection of chickens and the application of the molecular markers in screening chicken breeding materials resisting ALV-B infection are key points for realizing disease-resistant breeding of avian leukosis of subgroup B.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a molecular marker for resisting B subgroup avian leukosis virus infection of chickens, a screening/identifying method and application thereof, and the defects that the B subgroup avian leukemia occurrence and epidemic of chickens in China cannot be completely controlled by the prior art measures can be overcome.

The technical scheme for solving the technical problems is as follows: a method of screening/identifying avian leukosis subgroup B genetically resistant chickens is provided comprising: extracting genome DNA of a sample to be detected, amplifying tvb receptor gene fragments, sequencing, and judging whether the 3747 th position of the tvb receptor gene has deletion of a base G.

The method specifically comprises the following steps:

extracting DNA of a sample to be detected, amplifying tvb receptor gene fragments, and sequencing to judge whether the sample is a genetic anti-ALV-B infected chicken;

if the detection sample tvb receptor gene 3747 base position has homozygous deletion G mutation tvb ^delG/delG Indicating that the individual has a phenotype against ALV-B infection, the individual is a genetically anti-ALV-B infected chicken;

if no G deletion mutation occurs at 3747 th base position of tvb receptor gene in the detection sample, the genotype of the individual is indicated to be wild tvb ^s1/s1 The individual is an ALV-B susceptible chicken;

if the detection sample tvb receptor gene 3747 base position has heterozygous insertion G mutation tvb ^s1/delG The individual is ALV-B susceptible chicken, but the genotypes aretvb ^s1/delG The next generation generated after breeding the breeder cock and the breeder hen can generate the genotype of tvb ^delG/delG Thereby producing an individual resistant to ALV-B infection in a chicken.

Based on the technical scheme, the invention can also be improved as follows:

further, the amplification system used in the above amplification process comprises: 1. Mu.L of DNA template, 2.5. Mu.L of 10 Xbuffer, 2. Mu.L of dNTPs, 1. Mu.L of upstream and downstream detection primer, 0.5. Mu.L of KOD-FX, and finally ddH ₂ O was made up to 25. Mu.L.

Further, the amplification procedure used in the above amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 62℃for 30s, extension at 72℃for 30s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.

The method can be used for breeding the avian leukosis chicken variety/strain of the genetic anti-B subgroup.

The invention also provides a molecular marker for chicken genetic resistance to B subgroup avian leukosis virus infection, which is a base G deletion at 3747 th site of tvb receptor gene.

tvb receptor gene DNA sequences can be found in GenBank accession nos: NC_052553.1, which lacks base G at position 3747, abbreviated as tvb ^3747delG Mutation sites.

The invention also provides a primer for detecting the molecular marker, and the nucleotide sequence of the primer is as follows:

forward primer F:5'-GAGCCCGAGCAGAGCTGTGT-3' (SEQ ID NO: 1)

Reverse primer R:5'-CAGCTTCCAACTCACTCGG-3' (SEQ ID NO: 2).

The application of the molecular marker and/or the primer in screening/identifying the chicken infected by the avian leukosis virus of the genetic anti-subgroup B.

A kit for detecting/screening a genetic anti-B subgroup avian leukosis chicken, which comprises the primer.

The application of the kit in screening and breeding materials of the avian leukosis chicken variety/strain of the genetic anti-B subgroup.

The invention has the following beneficial effects:

the invention is divided intoThe genetic variation condition of tvb receptor genes of chicken breeds (comprising 1998 blood samples of 15 local chicken breeds and 15 yellow feather broiler strains) in China is analyzed, and the natural mutation of G deletion of 3747 th basic group of tvb receptor gene DNA sequence (GenBank accession number: NC_052553.1, specifically SEQ ID NO: 19) in chicken breeds in China is found, which is called tvb for short ^3747delG Mutation sites.

Based on the above study, tvb was then confirmed from two levels of in vitro and in vivo experiments ^3747delG Natural mutations cause infection of chickens against ALV-B. Of particular reason is tvb ^3747delG The mutation is located in the 4 th exon region of the tvb receptor gene, and the mutation is deleted G (tvb c.317 delG) at 317 th base of CDS (tvb receptor gene mRNA reference sequence NM_204115.3, see SEQ ID NO:20 in particular) of tvb receptor gene. Tvb c.317delG mutation results in a Tvb receptor protein sequence encoded thereby (Tvb receptor protein reference sequence NP 989446.2, see SEQ ID NO:21 in particular) beginning at amino acid residue 106 and ending encoding prematurely at amino acid residue 130, resulting in a change in the structure of the cysteine rich region (CRD) of the Tvb receptor protein (CRD of the Tvb receptor protein is the key domain mediating ALV-B infection and entry into host cells) and expression of a truncated Tvb ^delG Receptor proteins, thereby causing infection of the host against ALV-B.

The invention is also based on the molecular marker tvb of chicken genetic anti-B subgroup avian leukosis virus infection ^3747delG The established identification method for the chicken resistant to the ALV-B infection can quickly and accurately judge whether a detection sample is an ALV-B genetic resistance chicken or a susceptible chicken, can screen breeding materials for breeding the chicken variety/strain resistant to the ALV-B infection from chicken breeds (including local chicken breeds and commercial broiler chickens) in China, and provides technical support for breeding the chicken variety/strain resistant to the ALV-B infection.

Drawings

FIG. 1 shows a chicken breed tvb in China ^3747delG The result of the development and identification of mutation sites; wherein, FIG. 1A shows the PCR amplification result of the complete gene sequence of the chicken tvb receptor gene in China, M:2000marker, nc as negative control; 1-5 respectively represent the PCR amplification results of tvb gene 1, 2, 3, 4 and 5 gene fragments; FIG. 1B is a schematic diagram of the structure of the tvb receptor gene; drawing of the figureThe 1C red circle is marked as tvb ^3747delG At the mutation position of the tvb gene DNA sequence, the tvb receptor gene exon 4 3747 base is shown to generate G deletion mutation.

FIG. 2 is tvb ^3747delG Sequence analysis results of tvb gene coding region of mutation site.

FIG. 3 shows the results of the expression of mCherry-tvb fusion protein in 293-FT cells.

FIG. 4 shows construction of RCASBP (B) -EGFP recombinant expression plasmid and virus rescue results.

FIG. 5 is an RCASBP (B) -EGFP infection tvb ^3747delG Mutation site CEF process of different genotypes.

Detailed Description

The examples given below are only intended to illustrate the invention and are not intended to limit the scope thereof. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: tvb ^3747delG Development and identification of mutation sites

1. Primers designed for amplifying tvb receptor genes

Referring to the DNA sequence of chicken tvb receptor gene in NCBI database (GenBank accession number: NC_052553.1, see SEQ ID NO:19 specifically), 5 pairs of primers were designed to amplify the tvb gene full-length sequence 6596bp (1 segment, 2 segment, 3 segment, 4 segment and 5 segment) by 5-segment PCR, and the primer sequences, positions and PCR amplified segment sizes are shown in Table 1.

TABLE 1 full-length sequence PCR amplification primer information for tvb receptor genes

2. PCR amplification

Referring to the instruction book of the Tiangen blood DNA extraction kit, the genome DNA of 1998 blood samples of different chicken breeds (including 15 local chicken breeds and 15 yellow feather broiler breeds) in China is extracted, the 5 pairs of primers are used for PCR amplification of the full-length sequence of tvb receptor genes, and a PCR amplification system and an amplification program are shown in tables 2 and 3 respectively.

TABLE 2 PCR amplification System

TABLE 3 PCR amplification System

The PCR amplified products were detected by 2% agarose gel electrophoresis, and the results are shown in FIG. 1A. As can be seen from FIG. 1A, 5 fragments of 1079bp, 1254bp, 1530bp, 1239bp and 1451bp were amplified by PCR, and the sizes of the fragments were consistent with the sizes of the target fragments.

3. Sequencing analysis

The tvb receptor gene amplified product is sent to a biological engineering (Shanghai) stock company for purification and sequencing, DNAstar and Mutation Surveyor gene sequence analysis software is used for sequence comparison, genetic variation of chicken tvb receptor genes in China is analyzed, and ALV-B candidate genetic resistance sites are screened.

By analyzing the genetic variation of tvb receptor genes of 15 local chicken species and 15 yellow-feather broiler strains (1998 blood samples), G deletion of 3747 th base of exon 4 of tvb gene of Chinese chicken species was screened and found (tvb) ^3747delG ) Natural mutations of (a) (FIG. 1B). As shown in FIG. 1C, the sequence-sequencing profile is tvb from top to bottom ^3747delG Mutation site wild type tvb ^{s1 /s1} Individual, heterozygous mutant tvb ^s1/delG Individual and homozygous mutant tvb ^delG/delG The sequence of the individual, red line is marked as tvb acceptor gene sequence, and 3747 th base undergoes G deletion mutation.

Example 2: tvb ^3747delG Sequence analysis of coding region of tvb gene at mutation site

1. Extraction and reverse transcription of RNA

Acquisition tvb ^3747delG Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG 200 mu L of blood of the breeding hens is uniformly mixed in 800 mu L of TRIZOL (RNase Free EP tube) in an oscillating way, fully cracked, kept stand at room temperature for 5min, added with 300 mu L of chloroform, uniformly mixed in an oscillating way, centrifuged for 15min at 4 ℃ at 12,000Xg, the upper colorless transparent supernatant is sucked into a new EP tube, added with an equal volume of isopropanol solution to precipitate RNA, and kept stand for 10min; centrifuging at 4deg.C for 30min at 12,000Xg, discarding supernatant, washing precipitate with 75% ethanol once, discarding supernatant, standing at room temperature, and adding 50 μLDEPC water to dissolve RNA after ethanol is completely volatilized. To extract tvb ^3747delG Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG The blood RNA of the breeder was used as a template and was reverse transcribed into cDNA according to the instructions of the reverse transcription kit (Takara). The cDNA obtained by reverse transcription is preserved at-20 ℃ for standby.

2. RT-PCR amplification of tvb Gene full-Length coding region sequence

(1) Primer design

Referring to NCBI published tvb receptor gene mRNA sequence (NM_ 204115.3), primers were designed to amplify the entire coding region sequence (CDS) of tvb, and the primer sequences and RT-PCR amplified fragment sizes are shown in Table 4.

TABLE 4 RT-PCR amplification primer information for full-length coding region sequences of tvb receptor genes

(2) RT-PCR amplification

Tvb by ^3747delG Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG The cDNA of the chicken blood sample was used as a template for RT-PCR amplification with reference to Toyobo Co high-fidelity enzyme KOD-FX, and the amplification system and the amplification procedure are shown in Table 5 and Table 6, respectively.

TABLE 5 RT-PCR amplification System

TABLE 6 RT-PCR amplification System

After the amplification of the RT-PCR, the amplified product was detected by 1% agarose gel electrophoresis.

3. Clone sequencing analysis of RT-PCR amplified products

The recovery and purification of RT-PCR products were performed by reference to the procedures of the gel recovery and purification kit instruction of Axygen company. The RT-PCR purified product was ligated into pMD19-T Vector (Takara Co.) with reference to pMD19-T Vector. Referring to competent cell DH 5. Alpha. Instructions, ligation products were transformed into DH 5. Alpha. Competent cells. Positive colonies were screened, and PCR identified, and bacterial solutions identified as positive by PCR were sent to the division of bioengineering (Shanghai) for sequencing. Tvb was analyzed using Lasergene 7.0 software alignment ^3747delG Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG The sequence of the tvb gene full-length coding region is sequenced, and the result is shown in FIG. 2. FIG. 2A is a wild type tvb ^s1/s1 Homozygous mutant genotype tvb ^delG/delG RT-PCR amplification of the sequence of the tvb gene full-length coding region of the chicken blood sample, M:2000 markers; FIG. 2B is a partial nucleotide and deduced amino acid sequence of exon 4 surrounding a G single nucleotide deletion which would tvb ^r7 From tvb ^s1 (numbering from the beginning of the methionine codon). Red line tvb ^3747delG Amino acid sequence of G nucleotide deletion and alteration of mutation site; FIG. 2C is Tvb ^S1 And Tvb ^delG Schematic representation of the receptor protein. Tvb (Tvb) ^S1 Numbering of proteins includes Signal Peptide (SP), cysteine rich domains (CRD 1-3), transmembrane domain (MSD), and cytoplasmic death domain. At Tvb ^delG The position of the midamble shift mutation is highlighted with an asterisk, and at Tvb ^delG The premature stop codon TGA is indicated by a triangle. FIG. 2D is a deduced amino acid sequence of a comparison of known Tvb receptor CRDs. Presuming Tvb ^S1 Disulfide bonds in CRDs, tvb ^delG The putative cysteine substitutions in (1) are circled, tvb ^delG Amino acid residues of the frameshift peptide are shown in bold.

As can be seen from FIG. 2A, tvb ^3747delG Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG cDNA of breeding hens is taken as a template, and tvb is amplified by RT-PCR ^3747delG Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG A full length coding region sequence (CDS) band of the tvb gene, the band size being consistent with the size of the fragment of interest.

The RT-PCR amplified product clone sequencing analysis result shows that: tvb ^delG/delG G (tvb c.317 delG) was deleted from the 317 th base of the cDNA (FIG. 2B). tvb ^delG/delG G base deletion of cDNA sequence, resulting in Tvb encoded thereby ^delG The acceptor protein sequence was frameshifted from AA at position 106 and premature termination encoding occurred at AA at position 130 (fig. 2C). Thereby predicting tvb ^3747delG The frame shift mutation caused by the mutation causes the cysteine of the cysteine-rich region 3 (CRD 3) of the Tvb receptor protein to be replaced by other amino acid residues, resulting in the disulfide bond of the CRD3 to be broken, thereby changing the Tvb ^delG Structure of the receptor protein (fig. 2D).

Example 3: tvb ^3747delG Mutation results in Tvb gene expression of truncated Tvb ^delG Receptor proteins

1、pmCherry-C1-tvb ^s1/s1 、pmCherry-C1-tvb ^delG/delG Construction of recombinant expression plasmids

(1) RT-PCR amplification of tvb receptor gene mutation site full-length CDS fragment

RT-PCR amplification primers based on the sequence of the full-length coding region of the tvb receptor gene, xhoI and HindIII cleavage sites (underlined sequences) and protecting bases (as in Table 7) were added, respectively, and tvb was amplified by RT-PCR according to the method of example 2 ^{3215-3216insCC} Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG Tvb receptor gene full-length coding region sequence.

TABLE 7 RT-PCR amplification primers for full length CDS of tvb receptor gene

(2) Purification and ligation of PCR products

Reference was made to the Axygen gel recovery purification kit instructions for recovery of the purified RT-PCR amplification product and ligation to the pMD19-T vector.

(3) Ligation product transformation competent cells

Referring to competent cell DH 5. Alpha. Instructions, the ligation product was transformed into DH 5. Alpha. Competent cells.

(4) Double enzyme digestion reaction

Will contain tvb ^3747delG Mutation site wild type tvb ^s1/s1 And homozygous mutant tvb ^delG/delG the pMD19-T and pmcherry-C1 plasmids of the full-length coding region sequence fragment of the tvb gene were subjected to XhoI/HindIII double digestion respectively.

(5) Purification and ligation of the cleavage products

The target fragment and the pmcherry-C1 vector are recycled and purified according to the specification of the glue recycling and purifying kit of Axygen company, and then the target fragment and the pmcherry-C1 vector are uniformly mixed according to the ratio of 3:1, centrifuged and placed at 16 ℃ for overnight connection.

(6) Screening and identification of Positive colonies

Single white colonies are picked from the cultured plates, inoculated into 500 mu L Kana LB liquid medium, subjected to shaking culture at 37 ℃ for about 5 hours, directly subjected to PCR identification by taking bacterial liquid as a template, and sent to a biological engineering (Shanghai) stock company for sequencing.

(7) Restriction enzyme identification of pmCherry-C1-tvb recombinant plasmid

The recombinant plasmid of the positive bacterial liquid is extracted by referring to the instruction of a plasmid small-scale extraction kit (Edley). The pmCherry-C1-tvb recombinant plasmid was subjected to double restriction identification using the endonucleases XhoI and HindIII, and the recombinant plasmid was sent to the Probiotics (Shanghai) Co., ltd for sequencing verification, and the result is shown in FIG. 3. As can be seen from FIG. 3A, the recombinant expression plasmids were successfully constructed, and the constructed recombinant plasmids were designated as pmCherry-C1-tvb, respectively ^s1/s1 And pEGFPC1-tvb ^delG/delG 。

2. Western Blotting detection of pmCherry-C1-tvb recombinant expression plasmid

Referring to Lipofectamine 3000Reagent instructions, pmCher was performed using Lipofectamine 3000 transfection Reagentry-C1 control plasmid and pmCherry-C1-tvb ^s1/s1 、pEGFPC1-tvb ^delG/delG 293FT cells (6-well plates) prepared in advance and up to 70-80% full of monolayers were transfected respectively, and red fluorescence was observed after 24 hours. After 48h of transfection, 293FT cell supernatants were collected for Western Blotting detection, as shown in FIG. 3.

As can be seen from FIG. 3B, 24h after transfection, the transfection of pmCherry-C1, pmCherry-C1-tvb was detected under an inverted fluorescence microscope ^s1/s1 、pmCherry-C1-tvb ^delG/delG The recombinant plasmid 293-FT cells emit red fluorescence, and the result shows that mCherry-Tvb fusion protein is expressed in the 293-FT cells. 48h after transfection, cell supernatant samples were collected for Western Blotting detection, and the results showed that the blank control plasmid pmCherry-C1 expressed a protein of about 27kD in 293-FT cells, pmCherry-C1-tvb ^s1/s1 mCherry-Tvb expressed at about 70kD in 293-FT cells ^s1 Fusion protein, and pmCherry-C1-tvb ^delG/delG Recombinant plasmid expresses mCherry-Tvb with the size of about 41kD in 293-FT cells ^delG Fusion protein (FIG. 3C), results indicate tvb ^3747delG Mutation results in Tvb receptor gene expression of a truncated Tvb ^delG A receptor protein.

Example 4: tvb ^3747delG Identification of a mutation to render a host resistant to ALV-B infection

1. In vitro cell infection verification

(1) Construction of RCASBP (B) -EGFP recombinant expression plasmid, transfection of the recombinant expression plasmid into DF-1 cells, rescue and collection of RCASBP (B) -EGFP virus (namely, EGFP fluorescent protein ALV-B reporter virus) of DF-1 cell supernatant 7 days after transfection (FIG. 4, wherein A is a construction schematic diagram of RCASBP (B) -EGFP expression plasmid, B is restriction identification of RCASBP (B) -EGFP plasmid, C is RCASBP (B) -EGFP virus rescue), determination of virus Infection Unit (IU), split charging and storage at-80 ℃.

(2) Infection of tvb with ALV-B fluorescence reporter virus RCASBP (B) -EGFP, respectively ^3747delG Mutation site wild type tvb ^s1/s1 Heterozygous mutant tvb ^s1/delG And homozygous mutant tvb ^delG/delG CEF (chicken fibroblast CEF prepared from 9-day-old chick embryo hatched after breeding of the breeder examined in example 1), feelDetection of RCASBP (B) -EGFP virus infection tvb by flow cytometry 1, 2, 3, 7 days after dyeing ^3747delG The rate (%) of GPF positive cells indicates the infection rate of viruses in the case of CEF with different genotypes at the mutation sites, and the specific results are shown in FIG. 5.

FIG. 5A is RCASBP (B) -EGFP infection tvb ^s1/s1 、tvb ^s1/delG 、tvb ^delG/delG Time course of CEFs; FIG. 5B is a flow cytometry detection of RCASBP (B) -EGFP infection tvb ^3747delG Representative graph of GFP positive cell rate 7 days after CEFs of different genotypes at the mutation site. As can be seen from FIG. 5, RCASBP (B) -EGFP can efficiently infect wild type tvb ^s1/s1 CEFs, infection rate was approximately 20% on day 1, and almost all cells infected by day 7 (FIG. 5A). Whereas RCASBP (B) -EGFP pair tvb ^delG/delG CEFs showed a significant decrease in infection efficiency, with about 3% of the cells infected on day 1, and slow spread, about 11% of the cells infected on day 3, reaching 18% on day 7 (FIG. 5A). And tvb ^delG/delG In contrast, tvb ^s1/delG CEFs are more susceptible to infection by RCASBP (B) -EGFP, and exhibit similar infectious properties to the wild type (FIG. 5A). The GFP negative and GFP positive cells were significantly different, with two independent populations of GFP positive and negative cells in the FACS scatter plot (fig. 5B). The results show tvb ^3747delG The mutation causes infection of host cells in vitro against RCASBP (B) -EGFP.

2. In vivo toxicity attack test verification

(1) Will tvb ^3747delG Mutation sites wild-type, heterozygous mutant and homozygous mutant 1 day old chickens were randomly grouped, 25 animals per group, and kept in isolators. At 1 day of age, each chicken was inoculated intraperitoneally with an ALV-B SDAU09C2 strain virus solution (S/P value=2.2) 0.5ml, and at 5 days of age, challenged once again. 2 weeks after the virus challenge, an anticoagulated blood sample is collected from each chick, genomic DNA is extracted from each chick tvb by a direct sequencing method ^3747delG Genotyping was performed at the mutation site. 1 month after virus challenge, a blood sample of the chicken is collected, TRIZOL kit is used for extracting total RNA of the blood sample, ALV-BSDAU09C2 strain specificity detection primer is used for detecting viremia of each chicken by RT-PCR, and ALV-BSDAU09C2 strain infection tvb is determined ^3747delG Conditions of chicks of different genotypes were mutated.

Designing an upstream primer and a downstream primer for RT-PCR amplification of ALV-B-env:

env-F：5’-GCAGGCATTTCTGACTGGGC-3’(SEQ ID NO：17)；

env-R：5’-AGACCTTCCGATAGGTGAGG-3’(SEQ ID NO：18)。

(2) The env gene coding sequence of ALV-B is amplified by RT-PCR, and the length of the amplified fragment of the RT-PCR is 435bp. By means of

RT-PCR amplification was performed on One Step RT-PCR Kit Ver.2 Kit, PCR reaction procedure: reverse transcription is carried out for 30min at 50 ℃;94℃30s,58℃30s,72℃45s,35 cycles; extending for 10min at 72 ℃. The PCR products were detected by 2% agarose gel electrophoresis, if 435bp target band was observed, the sample developed viremia (ALV-B positive), if no target band was amplified, the sample did not develop viremia (ALV-B negative), and the results are shown in Table 8.

TABLE 8 ALV-B infection tvb ^3747delG Post-viremia positive condition of chickens with different mutation sites

As shown in Table 8, 26 wild-type tvb were 1 day old ^s1/s1 The chicks were all ALV-B positive after inoculation with ALV-B SDAU09C2 strain virus solution. And 27 days old of 1 day tvb ^delG/delG After the genetic chicks are inoculated with the ALV-B SDAU09C2 strain virus liquid, the ALV-B positive rate is reduced to 7.4 percent, and 29 chicks are tvb at 1 day old ^s1/delG The ALV-B positive rate of the genotype chicks after the chicks were inoculated with the virus liquid of the ALV-B SDAU09C2 strain was 90.9% (Table 8). The toxicity attack test result shows that the ALV-B in-vivo infection is consistent with the in-vitro infection result, and tvb is proved ^3747delG Mutant host chicken against ALV-B infection, tvb ^3747delG Naturally mutating into a molecular marker for resisting ALV-B infection of chicken.

Example 5: establishment and application of identification standard for chicken resisting ALV-B infection

1. Establishment of identification standards for ALV-B infection resistant chickens

(1) Primer design

With reference to the tvb receptor gene DNA sequence (GenBank accession number: NC-052553.1), PCR primers (forward primer F:5'-GAGCCCGAGCAGAGCTGTGT-3' (SEQ ID NO: 1) and reverse primer R:5'-CAGCTTCCAACTCACTCGG-3' (SEQ ID NO: 2)) were designed to amplify the primer comprising tvb ^3747delG Tvb receptor genomic region of the mutation site.

(2)tvb ^3747delG PCR amplification of the tvb receptor genomic region at the mutation site

The PCR reaction system comprises: 1. Mu.L of DNA template, 2.5. Mu.L of 10 Xbuffer, 2. Mu.L of dNTPs, 1. Mu.L of upstream and downstream detection primer (the nucleotide sequences of which are shown as SEQ ID NO:1 and SEQ ID NO: 2), 0.5. Mu.L of KOD-FX, ddH ₂ O was made up to 25. Mu.L.

PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 62℃for 30s, extension at 72℃for 30s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.

(3) Sequencing of PCR amplified products

The PCR amplified product was detected by 2% agarose gel electrophoresis, purified and sequenced by Shanghai Bioengineering (Shanghai) Co., ltd, and tvb was obtained according to the sequencing result ^3747delG Genotyping was performed at the mutation site.

According to tvb ^3747delG The mutation site genotypes establish the identification criteria for ALV-B-infected chickens, as shown in Table 9.

TABLE 9 identification criteria for anti-ALV-B infected chickens

If tvb ^3747delG The genotype of the resistance locus is wild type tvb ^s1/s1 No resistance to ALV-B infection (susceptibility), the individual being an ALV-B susceptible chicken;

if tvb ^3747delG The resistance locus genotype was tvb ^s1/delG Susceptible to infection by ALV-B, but the individual carries an ALV-B genetic resistance recessive gene;

if tvb ^3747delG Resistance siteGenotype tvb ^delG/delG Genetic resistance is then developed to the infection with ALV-B and the individual is a chicken resistant to the infection with ALV-B.

2. Application of chicken identification standard for resisting ALV-B infection

(1) Extraction of whole genome DNA

According to the instruction of the extraction kit of the root blood genome, the DNA of the anticoagulated blood of 15 local chicken species in China and 15 yellow-feather broiler strains is extracted and stored at the temperature of minus 20 ℃ for standby.

(2) PCR amplification and sequencing

The PCR amplification primers and the amplification procedure are as described in examples 5 and 1, for different chicken species tvb in China ^3747delG And (3) carrying out PCR amplification on the tvb genome region of the resistance locus, and sequencing the PCR amplification product after the PCR amplification product is detected to be qualified by 2% agarose gel electrophoresis and then sent to a biological engineering (Shanghai) Co., ltd.

(3)tvb ^3747delG Resistance locus genotyping

Sequencing peak pattern analysis of sequencing results using Chromas software, sequencing sample tvb was analyzed ^3747delG Genotype of the resistance locus.

Chinese local chicken species and yellow-feather broiler strain tvb ^3747delG The genotyping results of the resistance loci are shown in Table 10, and tvb are present in local chickens such as Beijing oil chickens, ningdu yellow chickens, tibetan chickens and the like ^3747delG Resistance genotype tvb of resistance locus ^delG/delG The frequencies of the yellow-feather broiler strain 2, the yellow-feather broiler strain 6, the yellow-feather broiler strain 10, the yellow-feather broiler strain 19 and the yellow-feather broiler strain 20 are 0.17, 0.08 and 0.15 respectively, and tvb also exist ^3747delG Resistance genotype tvb of resistance locus ^delG/delG The frequencies of the breeding materials are respectively 0.13, 0.15, 0.13, 0.17 and 0.06, which shows that the local chicken species and yellow feather broiler chicken strains have good anti-ALV-B genetic improvement potential, and the breeding materials for resisting the ALV-B infection can be screened from the chicken species and can be used for breeding chicken varieties (strains) resisting the ALV-B infection so as to prevent and control B subgroup avian leukosis.

TABLE 10 China chicken breeds tvb ^3747delG Genotype frequency distribution of resistance loci

All nucleotide sequences in this application are as in Table 11 (wherein SEQ ID NO:19 is not described herein):

table 11 sequence listing

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (10)

1. A method of screening/identifying avian leukosis subgroup B genetically resistant chickens comprising: extracting genome DNA of a sample to be detected, amplifying tvb receptor gene fragments, sequencing, and judging whether the 3747 th position of the tvb receptor gene has deletion of a base G.

2. The method of claim 1, wherein the presence of the homozygous deletion G mutation tvb at base position 3747 of the receptor gene of sample tvb is detected ^delG/delG The individual is a genetically anti-ALV-B infected chicken;

if the detected sample tvb receptor gene 3747 base position does not have G deletion mutation, the individual is ALV-B susceptible chicken;

if the detection sample tvb receptor gene 3747 base position has heterozygous insertion G mutation tvb ^s1/delG The individual is an ALV-B susceptible chicken, but it carries an ALV-B genetic resistance recessive gene.

3. According to claim 1The method is characterized in that an amplification system used in the amplification process comprises: 1. Mu.L of DNA template, 2.5. Mu.L of 10 Xbuffer, 2. Mu.L of dNTPs, 1. Mu.L of upstream and downstream detection primer, 0.5. Mu.L of KOD-FX, and finally ddH ₂ O was made up to 25. Mu.L.

4. The method of claim 1, wherein the amplification procedure used in the amplification process is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 62℃for 30s, extension at 72℃for 30s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.

5. Use of the method according to any one of claims 1-4 for breeding a genetically anti-subgroup B avian leukosis chicken variety/strain.

6. A molecular marker for chicken genetic resistance to infection by avian leukosis virus subgroup B, which is characterized in that the molecular marker is a base G deletion at 3747 of tvb receptor gene.

7. A primer for detecting a molecular marker of avian leukosis virus infection of subgroup B of chicken genetically against as claimed in claim 6, wherein the nucleotide sequence of the primer is as follows:

forward primer F:5'-GAGCCCGAGCAGAGCTGTGT-3';

reverse primer R:5'-CAGCTTCCAACTCACTCGG-3'.

8. Use of the molecular marker of the chicken genetic anti-subgroup B avian leukosis virus infection of claim 6 and/or the primer of claim 7 for screening/identifying the chicken genetic anti-subgroup B avian leukemia virus infection.

9. A kit for detecting/screening avian leukosis of subgroup B genetically directed against chickens, comprising the primer of claim 7.

10. The use of the kit of claim 9 for screening breeding materials of a variety/strain of a avian leukosis-resistant subgroup B genetic subgroup B chicken.

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