WO2023193140A1 - 牛结节性皮肤病病毒lsdv/ch/jy/2021的全基因组序列及其扩增引物 - Google Patents

牛结节性皮肤病病毒lsdv/ch/jy/2021的全基因组序列及其扩增引物 Download PDF

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WO2023193140A1
WO2023193140A1 PCT/CN2022/085327 CN2022085327W WO2023193140A1 WO 2023193140 A1 WO2023193140 A1 WO 2023193140A1 CN 2022085327 W CN2022085327 W CN 2022085327W WO 2023193140 A1 WO2023193140 A1 WO 2023193140A1
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nucleotide sequence
seq
sequence
primer
sequence listing
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王鹏
辛俊利
黄海碧
宋庆庆
赵丽霞
刘玉梅
刘建奇
刘东霞
张彦婷
范文霞
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金宇保灵生物药品有限公司
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Priority to PCT/CN2022/085327 priority patent/WO2023193140A1/zh
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  • the invention belongs to the field of biological technology, and specifically relates to the full genome sequence of a bovine nodular skin disease virus (named: LSDV/CH/JY/2021) and its amplification primers.
  • Lumpy Skin Disease Virus is an enveloped double-stranded DNA virus of the genus Sheeppoxvirus in the family Poxviridae. The other two viruses in this genus are Sheeppox Virus (SPV). ) and goatpox virus (Goatpox Virus, GTPV). LSDV can cause bovine nodular dermatosis, which is an acute, subacute or chronic infectious disease characterized by extensive nodules on the skin, mucous membranes and organ surfaces. It often manifests as fever and swollen lymph nodes on the body surface of sick cows, and can occur in lactating cows.
  • SPV Sheeppox Virus
  • GTPV goatpox virus
  • LSDV The genome size of LSDV is approximately 15,100 bp, consisting of a central coding region and 2.2 kb inverted terminal repeats at both ends, encoding 157 predicted genes. Compared with poxviruses of this genus or other genera, LSDV has 146 conserved genes. In the central genomic region, the LSDV gene shares about 65% genetic homology with swine poxvirus and capripoxvirus. In the terminal region, the collinearity is destroyed, and the homology between the LSDV gene and the other two poxvirus genes is about 43%. LSDV is genetically and structurally similar to herpesviruses, but it contains IL-10, IL-1 binding protein, G protein-coupled CC chemokine receptor, and surface growth factor-like protein homologs of other poxvirus genera.
  • the LSDV virus was discovered in a cattle farm in Guangzhou City, Guangdong province, China.
  • One of the LSDV viruses isolated by the inventor from diseased cattle material was named Lumpy Skin Disease Virus LSDV/CH/JY/2021.
  • Bovine nodular skin disease virus LSDV/CH/JY/2021 strain can be cultured and proliferated on bovine kidney cells (MDBK cells), African green monkey kidney cells (Vero) and goat kidney cells. According to the occurrence of CPE in MDBK cells of the LSDV/CH/JY/2021 strain, the infection dose of half of the virus tissue cells (TCID 50 ) was determined to be greater than or equal to 10 5.5 TCID 50 /mL.
  • the full genome sequence of the virus is the basis for studying the pathogenicity and pathogenesis of the virus, developing diagnostic reagents, and developing vaccines. Therefore, the acquisition of the full genome sequence of the bovine nodular skin disease virus LSDV/CH/JY/2021 is important for further research and utilization. This strain is of great significance.
  • one aspect of the present invention provides a complete genome sequence of a bovine nodular skin disease virus (named: LSDV/CH/JY/2021), and its nucleotide sequence is as follows: The nucleotide sequence shown in SEQ ID NO:1 in the list, or the nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing under high stringency conditions.
  • the 336bp-2527bp from the 5' end and the 148415bp-150606bp from the 5' end are palindromic inverted repeats, and the 9bp-150599bp from the 5' end are coding (CDS) sequence.
  • Another aspect of the present invention provides the complete genome sequence of bovine nodular skin disease virus LSDV/CH/JY/2021 and the protein sequence encoded by its coding (CDS) sequence.
  • amino acid sequence of the above protein sequence is shown as SEQ ID NO: 2 in the sequence listing.
  • the present invention also provides a primer combination for PCR amplification of the complete genome sequence of bovine nodular skin disease virus, which consists of 30 pairs of primers, respectively used to amplify the complete genome sequence of bovine nodular skin disease virus.
  • 30 nucleotide sequence fragments S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21 , S22, S23, S24, S25, S26, S27, S28, S29 and S30
  • the primer combination includes:
  • the primer pair used for PCR amplification of the S1 nucleotide sequence fragment is as shown in SEQ ID NO: 3 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO:4 shown;
  • the primer pair used for PCR amplification of the S2 nucleotide sequence fragment is as shown in SEQ ID NO: 5 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:6 shown;
  • the primer pair used for PCR amplification of the S3 nucleotide sequence fragment is as shown in SEQ ID NO: 7 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing.
  • SEQ ID NO: 8 is shown;
  • the primer pair used for PCR amplification of the S4 nucleotide sequence fragment is as shown in SEQ ID NO: 9 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO:10 shown;
  • the primer pair used for PCR amplification of the S5 nucleotide sequence fragment is as shown in SEQ ID NO: 11 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:12 shown;
  • the primer pair used for PCR amplification of the S6 nucleotide sequence fragment is as shown in SEQ ID NO: 13 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:14 shown;
  • the primer pair used for PCR amplification of the S7 nucleotide sequence fragment is as shown in SEQ ID NO: 15 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO:16 shown;
  • the primer pair used for PCR amplification of the S8 nucleotide sequence fragment is as shown in SEQ ID NO: 17 in the sequence listing, and the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO:18 shown;
  • the primer pair used for PCR amplification of the S9 nucleotide sequence fragment is as shown in SEQ ID NO: 19 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing.
  • SEQ ID NO: 20 is shown;
  • the primer pair used for PCR amplification of the S10 nucleotide sequence fragment is as shown in SEQ ID NO: 21 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:22 shown;
  • the primer pair used for PCR amplification of the S11 nucleotide sequence fragment is as shown in SEQ ID NO: 23 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 24 shown;
  • the primer pair used for PCR amplification of the S12 nucleotide sequence fragment is as shown in SEQ ID NO: 25 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO: 26 shown;
  • the primer pair used for PCR amplification of the S13 nucleotide sequence fragment is as shown in SEQ ID NO: 27 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 28 shown;
  • the primer pair used for PCR amplification of the S14 nucleotide sequence fragment is as shown in SEQ ID NO: 29 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 30 is shown;
  • the primer pair used for PCR amplification of the S15 nucleotide sequence fragment is as shown in SEQ ID NO: 31 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:32 shown;
  • the primer pair used for PCR amplification of the S16 nucleotide sequence fragment is as shown in SEQ ID NO: 33 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 34 shown;
  • the primer pair used for PCR amplification of the S17 nucleotide sequence fragment is as shown in SEQ ID NO: 35 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 36 shown;
  • the primer pair used for PCR amplification of the S18 nucleotide sequence fragment is as shown in SEQ ID NO: 37 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 38 shown;
  • the primer pair used for PCR amplification of the S19 nucleotide sequence fragment is as shown in SEQ ID NO: 39 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing.
  • SEQ ID NO: 40 is shown;
  • the primer pair used for PCR amplification of the S20 nucleotide sequence fragment is as shown in SEQ ID NO: 41 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 42 shown;
  • the primer pair used for PCR amplification of the S21 nucleotide sequence fragment is as shown in SEQ ID NO: 43 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO: 44 shown;
  • the primer pair used for PCR amplification of the S22 nucleotide sequence fragment is as shown in SEQ ID NO: 45 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 46 shown;
  • the primer pair used for PCR amplification of the S23 nucleotide sequence fragment is as shown in SEQ ID NO: 47 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO: 48 shown;
  • the primer pair used for PCR amplification of the S24 nucleotide sequence fragment is as shown in SEQ ID NO: 49 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:50 shown;
  • the primer pair used for PCR amplification of the S25 nucleotide sequence fragment is as shown in SEQ ID NO: 51 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:52 shown;
  • the primer pair used for PCR amplification of the S26 nucleotide sequence fragment is as shown in SEQ ID NO: 53 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing.
  • the primer pair used for PCR amplification of the S27 nucleotide sequence fragment is as shown in SEQ ID NO: 55 in the sequence listing, and the nucleotide sequence of its downstream primer is as shown in the sequence listing. SEQ ID NO:56 shown;
  • the primer pair used for PCR amplification of the S28 nucleotide sequence fragment is as shown in SEQ ID NO: 57 in the sequence listing, and the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO:58 shown;
  • the primer pair used for PCR amplification of the S29 nucleotide sequence fragment is as shown in SEQ ID NO: 59 in the sequence listing.
  • the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO:60 shown;
  • the primer pair used for PCR amplification of the S30 nucleotide sequence fragment is as shown in SEQ ID NO: 61 in the sequence listing, and the nucleotide sequence of the downstream primer is as shown in the sequence listing. SEQ ID NO:62 is shown.
  • the primer combination provided by the present invention for PCR amplification of the complete genome sequence of bovine nodular skin disease virus can be used for any known bovine nodular skin disease virus, or the bovine nodular skin disease virus involved in the present invention.
  • Another aspect of the present invention also provides a method for obtaining the complete genome sequence of bovine nodular skin disease virus, which includes the following steps:
  • step 5 Perform high-throughput sequencing on the total DNA of the bovine nodular skin disease virus extracted in step 1), and splice, edit and correct the obtained high-throughput sequencing raw data according to the reference sequence and adapter primers to obtain the second Initial whole-genome sequence;
  • the method for obtaining the complete genome sequence of bovine nodular skin disease virus provided by the present invention can be used for any known bovine nodular skin disease virus, or the bovine nodular skin disease virus LSDV/ CH/JY/2021.
  • the reference sequence in step 5) is any one or several of the sequences represented by the following GenBank accession numbers: NC_003027.1, MN072619.1, KX683219.1, MW631933.1, AF325528.1 , AF409137.1, MW656253.1, KX894508.1, MN995838.1, MH893760.2, MW699032.1, MN642592.1, MT130502.2, KY829023.3, MT643825.1, KY702007.1, MT992618.1 ,MT134042 .1, MH646674.1, MW435866.1, MK441838.1, KX764645.1, AF409138.1, KX764643.1, KX764644.1, MG972412.1, MW656252.1, MN636842.1, MN636840.1, MN636 838.1 , MN636841.1, MN636843.1, MN636843.1,
  • the nucleotide sequence of the adapter primer described in step 5 is as shown in SEQ ID NO: 63 and SEQ ID NO: 64 in the sequence listing.
  • the reaction system (50 ⁇ L) of the PCR method in step 2) includes: 1-2 ⁇ L of total DNA, 20-30 ⁇ L of KOD One TM PCR Master Mix, 1-2 ⁇ L of upstream primer (10 ⁇ M), and 1-2 ⁇ L of downstream primer ( 10 ⁇ M) 1-2 ⁇ L, Nuclease-free Water 15-25 ⁇ L.
  • the reaction program of the PCR method in step 2) includes: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 10s, annealing at 50-60°C for 30s, extension at 68°C for 30s-360s, a total of 35 cycles; and finally Extension at 68°C for 10 minutes.
  • the full genome sequence of bovine nodular skin disease virus LSDV/CH/JY/2021 and its amplification primers are provided.
  • the primers used for PCR amplification of the complete genome sequence of bovine nodular skin disease virus provided by the invention are composed of 30 pairs of primers, and these primer pairs can be used to amplify bovine nodular skin disease virus (which may include LSDV/CH/JY/ 2021) 30 nucleotide sequence fragments (S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20 , S21, S22, S23, S24, S25, S26, S27, S28, S29 and S30), after splicing, editing and correcting the overlapping DNA sequences of the 30 amplified nucleotide sequence fragments, use them to complement
  • the bovine nodular skin disease virus sequence can be accurately
  • bovine nodular skin disease virus LSDV/CH/JY/2021 can include the full genome sequence of LSDV/CH/JY/2021.
  • the whole genome sequence of bovine nodular skin disease virus LSDV/CH/JY/2021 provided by the present invention is beneficial to the pathogenesis, molecular epidemiology and genetics of bovine nodular skin disease virus LSDV/CH/JY/2021. Further research can lay important data support and theoretical foundation for the development of diagnostic reagents and vaccines for bovine nodular skin disease virus LSDV/CH/JY/2021.
  • Figure 1 shows the 30 nucleotide sequence fragments of the complete genome of bovine nodular skin disease virus LSDV/CH/JY/2021 (S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11 , S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, S29 and S30), 1.0% agarose gelation Gel electrophoresis pattern; Panel A shows 1.0 of the PCR amplification products of S1, S2, S4, S5, S6, S8, S9, S11, S12, S13, S15, S17, S19, S21, S23, S25 and S29 % agarose gel electrophoresis pattern.
  • Panel B shows the 1.0% agarose gel electrophoresis pattern of the PCR amplification products of S3, S14, S20, S24, S28 and S30.
  • Panel C shows the PCR amplification products of S10 and S16. 1.0% agarose gel electrophoresis pattern of the amplification product.
  • Panel D shows the 1.0% agarose gel electrophoresis pattern of the PCR amplification product of S18.
  • Panel E shows the PCR amplification products of S7, S17, S21 and S22.
  • Panel F shows the 1.0% agarose gel electrophoresis pattern of the PCR amplification products of S26, S27 and S30.
  • Example 1 Design of primers for PCR amplification of 30 nucleotide sequence fragments of the whole genome of bovine nodular skin disease virus LSDV/CH/JY/2021
  • AH010683.2, MW732649.1, MZ577075.1, MZ577073.1, MZ577076.1, MZ577074.1 and MW355944.1 were analyzed, compared, screened and optimized using bioinformatics DNAStar software, and the final determination of the cattle
  • the full genome sequence of nodular dermatosis virus LSDV/CH/JY/2021 is divided into 30 nucleotide sequence fragments: S1 (485bp), S2 (1471bp), S3 (432bp), S4 (9274bp), S5 (7408bp) , S6(5941bp), S7(13325bp), S8(6727bp), S9(5492bp), S10(947bp), S11(9845bp), S12(8650bp), S13(6747bp), S14(445bp), S15(5291bp) , S16(1255bp), S17(9647bp), S18
  • Example 2 Obtaining the full genome sequence of bovine nodular skin disease virus LSDV/CH/JY/2021
  • This example performs PCR amplification of the total DNA of bovine nodular skin disease virus LSDV/CH/JY/2021 based on the primers obtained in Example 1, and finally obtains the complete DNA of bovine nodular skin disease virus LSDV/CH/JY/2021.
  • Genome sequence specifically including the following steps:
  • the total DNA of bovine nodular skin disease virus LSDV/CH/JY/2021 was extracted according to the instructions of AxyprepTM Body Fluid Viral DNA/RNA Miniprep Kit (purchased from AXYGEN Company). The specific steps are as follows:
  • Reagent preparation Prepare isopropyl alcohol containing 1% (V/V) glacial acetic acid in advance; add the specified volume of absolute ethanol to reagent Buffer W1A and Buffer W2 respectively. That is, add 17mL absolute ethanol to 24mL Buffer W1A; add 56mL absolute ethanol to 24mL Buffer W2.
  • venom sample (venom obtained by culturing bovine nodular skin disease virus LSDV/CH/JY/2021 on bovine kidney cells (MDBK)), and determine that the viral half tissue cell infection dose (TCID 50 ) is greater than 10 5.5 TCID 50 /mL; the venom is obtained by: inoculating bovine nodular skin disease virus LSDV/CH/JY/2021 to expand the culture of MDBK cells, and culture the cells in a spinner flask for about 12 days until 70% of the area falls off and appears as a mesh. Collect poison) into 1.5mL centrifuge tube. Repeat freezing and thawing three times, centrifuge at 10,000 ⁇ g for 15 min, and collect the supernatant; centrifuge at 22,000 ⁇ g and 4°C for 2 h, discard the supernatant, and collect the precipitate.
  • MDBK bovine kidney cells
  • the total DNA of bovine nodular skin disease virus LSDV/CH/JY/2021 extracted in step 1) was used as a template, and under the guidance of the primers (shown in Table 1) provided in Example 1, the bovine nodular skin disease virus LSDV/CH/JY/2021 was amplified by PCR.
  • Table 2 Reaction system for PCR amplification of 30 nucleotide sequence fragments of the entire genome of bovine nodular skin disease virus LSDV/CH/JY/2021
  • Table 3 Reaction procedure for PCR amplification of 30 nucleotide sequence fragments of the entire genome of bovine nodular skin disease virus LSDV/CH/JY/2021
  • short fragments refer to fragments with a length of less than 2000 bp
  • long fragments refer to fragments with a length of more than 2000 bp.
  • Reagent preparation Add the specified volume of absolute ethanol to the Wash Buffer (specifically, add 45 mL of absolute ethanol to 9 mL).
  • Table 4 Use Reaction system for connecting 3' and 5' target nucleotide sequence fragments with vector
  • Transformation Transform the ligation products of the 3' and 5' target nucleotide sequence fragments (S1 and S30) into E. Coli JM109 competent cells (purchased from TaKaRa Company). The specific steps are as follows:
  • step (3.5) Take 200 ⁇ L of the bacterial liquid in step (3.4), transfer it to an LB agar plate containing 50 ⁇ g/mL ampicillin, and spread the plate evenly. After all the bacterial liquid is absorbed, incubate upside down in a 37°C incubator for 12-14 hours.
  • the second initial whole-genome sequence obtained by second-generation sequencing technology may be incomplete and lack some small gene fragments, especially the 3' of the whole-genome sequence) end and 5' end sequences, so it is necessary to use the first initial whole genome sequence amplified by 30 pairs of primers to complete the second initial whole genome sequence), and the second initial whole genome sequence after completion is used as the bovine nodule
  • the complete genome sequence of the sexually transmitted skin disease virus LSDV/CH/JY/2021, its nucleotide sequence is shown as SEQ ID NO: 1 in the sequence listing.
  • SEQ ID NO: 1 shows that the full genome sequence of bovine nodular skin disease virus LSDV/CH/JY/2021 consists of 150606 nucleotides, starting from 336bp-2527bp at the 5' end ( 2192bp) and the 148415bp-150606bp (2192bp) from the 5' end are palindromic inverted repeats of each other; starting from the 9bp-371bp from the 5' end is the first coding (CDS) sequence, and the 150120bp-150599bp from the 5' end is The 157th coding (CDS) sequence, that is, the nucleotide (DNA) sequence of the coding (CDS) sequence of bovine nodular skin disease virus LSDV/CH/JY/2021 predicted based on the whole genome sequence is SEQ ID NO: 1 It is shown from 9bp to 150599bp from the 5' end, and is 150591bp long.
  • bovine nodular skin disease virus LSDV/CH/JY/2021 can also be used to refer to existing databases (such as NR, SwissProt, Pfam, COG_category , GO, KEGG_pathway, PHI and TCDB database) to compare the coding sequence (CDS) map of bovine nodular skin disease virus LSDV/CH/JY/2021.
  • existing databases such as NR, SwissProt, Pfam, COG_category , GO, KEGG_pathway, PHI and TCDB database
  • CDS protein sequence encoded by the coding sequence (CDS) of bovine nodular skin disease virus LSDV/CH/JY/2021 is as follows: SEQ ID NO in the sequence listing As shown in :2, SEQ ID NO:2 in the sequence list is obtained by encoding 157 coding sequences (CDS).
  • KY702007. 1. KX894508.1, MN995838.1, MH893760.2, MW699032.1, MN642592.1, MT130502.2, KY829023.3, MT643825.1, MT992618.1, MT134042.1, MH646674.1, AH010683. 2.
  • a genetic evolutionary tree was constructed from the whole genome sequences of MN636840.1, MW732649.1, MZ577075.1, MZ577073.1, MZ577076.1, MZ577074.1, MW355944.1, MK860688.1), and the results showed that bovine nodular skin
  • the virus LSDV/CH/JY/2021 is located in the same branch as the reference strain of the LSDV virus species, indicating that LSDV/CH/JY/2021 belongs to the LSDV virus species, and proving that LSDV/CH/JY/2021 is the same as China/GD01/2020 (MW355944.1) has the highest homology.
  • the invention provides the full genome sequence of the bovine nodular skin disease virus LSDV/CH/JY/2021 and its amplification primers.
  • the full genome sequence of the bovine nodular skin disease virus LSDV/CH/JY/2021 can accurately reflect the bovine nodular skin disease virus LSDV/CH/JY/2021.
  • the genome information of the dermatosis virus LSDV/CH/JY/2021 will be used to facilitate research on the pathogenesis of the virus, as well as the development of diagnostic reagents and vaccines for the virus.

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Abstract

牛结节皮肤病病毒LSDV/CH/JY/2021的全基因组序列及其扩增引物。提供的用于PCR扩增牛结节性皮肤病病毒的全基因组序列的引物由30对引物组成，将这些引物对扩增获得的30个核苷酸序列片段依次进行拼接、编辑及校正，并用其补全用二代测序方法测得的牛结节皮肤病病毒的核苷酸序列片段，得到牛结节皮肤病病毒的全基因组序列。提供的牛结节皮肤病LSDV/CH/JY/2021的全基因组序列有利于该病毒的致病机制等的研究，并有利于针对该病毒的诊断试剂开发和疫苗研制等。

Description

牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列及其扩增引物 技术领域
本发明属于生物学技术领域，具体涉及一种牛结节性皮肤病病毒(命名为：LSDV/CH/JY/2021)的全基因组序列及其扩增引物。
背景技术
牛结节性皮肤病病毒(Lumpy Skin Disease Virus，LSDV)是痘病毒科羊痘病毒属的一种有囊膜的双链DNA病毒，该属另外两种病毒是绵羊痘病毒(Sheeppox Virus，SPV)和山羊痘病毒(Goatpox Virus，GTPV)。LSDV能引起牛结节皮肤病，是一种皮肤黏膜、器官表面广泛性结节为特征的急性、亚急性或慢性传染病，常表现为病牛发热，体表淋巴结肿大，泌乳牛可发生乳房炎，公牛睾丸炎造成永久或暂时性不育等，严重时导致病牛死亡。这给我国的奶牛和肉牛养殖造成了严重的经济损失。并且LSDV作为一种新出现的病毒，其潜在影响是不可估量的，必须足够重视。
LSDV的基因组大小约为15100bp，是由中央的编码区和两端长为2.2kb的倒置末端重复序列组成，编码157个预测基因。LSDV与该属或其他属的痘病毒比较，有146个保守基因。在中央基因组区域，LSDV基因与猪痘病毒和山羊痘病毒病毒的基因同源性为65％左右。在末端区域，共线性被破坏，LSDV基因同其他两种痘病毒基因同源性在43％左右。LSDV在基因和病毒结构上与疱疹病毒相似，但它含有其他痘病毒属的IL-10、IL-1结合蛋白、G蛋白偶联CC趋化因子受体和表面生长因子样蛋白同源物。
在中国广东省广州市某牛场发现了LSDV病毒，发明人从发病牛只病料分离得到的LSDV病毒中一株被命名为Lumpy Skin Disease Virus LSDV/CH/JY/2021。牛结节性皮肤病病毒LSDV/CH/JY/2021株可在牛肾细胞(MDBK细胞)、非洲绿猴肾细胞(Vero)和山羊肾细胞上培养增殖。根据LSDV/CH/JY/2021株在MDBK细胞出现CPE情况，测定其病毒半数组织细胞感染量(TCID 50)大于等于10 5.5TCID 50/mL。病毒全基因组序列是研究病毒的致病性、致病机制、开发诊断试剂和研制疫苗的基础，因此牛结节性皮肤病病毒LSDV/CH/JY/2021全基因组序列的获得对进一步研究和利用该病毒株具有重要意义。
发明内容
针对现有技术中存在的一个或多个问题，本发明一个方面提供一种牛结节皮肤病病毒(命名为：LSDV/CH/JY/2021)的全基因组序列，其核苷酸序列如序列表中SEQ ID NO:1所示，或在高严谨条件下可与序列表中SEQ ID NO:1所示的核苷酸序列杂交的核苷 酸序列。
在SEQ ID NO:1所示的核苷酸序列中，自5’端第336bp-2527bp与自5’端第148415bp-150606bp互为回文结构倒置重复，自5’端第9bp-150599bp为编码(CDS)序列。
本发明另一方面提供了牛结节皮肤病病毒LSDV/CH/JY/2021的全基因组序列及其编码(CDS)序列编码的蛋白序列。
上述蛋白序列的氨基酸序列如序列表中SEQ ID NO：2所示。
本发明再一方面还提供了用于PCR扩增牛结节性皮肤病病毒的全基因组序列的引物组合，其由30对引物组成，分别用于扩增牛结节性皮肤病病毒的全基因组的30个核苷酸序列片段：S1、S2、S3、S4、S5、S6、S7、S8、S9、S10、S11、S12、S13、S14、S15、S16、S17、S18、S19、S20、S21、S22、S23、S24、S25、S26、S27、S28、S29和S30，所述引物组合包括：
(1)用于PCR扩增S1核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：3所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：4所示；
(2)用于PCR扩增S2核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：5所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：6所示；
(3)用于PCR扩增S3核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：7所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：8所示；
(4)用于PCR扩增S4核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：9所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：10所示；
(5)用于PCR扩增S5核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：11所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：12所示；
(6)用于PCR扩增S6核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：13所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：14所示；
(7)用于PCR扩增S7核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：15所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：16所示；
(8)用于PCR扩增S8核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：17所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：18所示；
(9)用于PCR扩增S9核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：19所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：20所示；
(10)用于PCR扩增S10核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：21所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：22所示；
(11)用于PCR扩增S11核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：23所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：24所示；
(12)用于PCR扩增S12核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：25所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：26所示；
(13)用于PCR扩增S13核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：27所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：28所示；
(14)用于PCR扩增S14核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：29所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：30所示；
(15)用于PCR扩增S15核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：31所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：32所示；
(16)用于PCR扩增S16核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：33所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：34所示；
(17)用于PCR扩增S17核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：35所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：36所示；
(18)用于PCR扩增S18核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：37所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：38所示；
(19)用于PCR扩增S19核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：39所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：40所示；
(20)用于PCR扩增S20核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：41所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：42所示；
(21)用于PCR扩增S21核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：43所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：44所示；
(22)用于PCR扩增S22核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：45所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：46所示；
(23)用于PCR扩增S23核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：47所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：48所示；
(24)用于PCR扩增S24核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：49所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：50所示；
(25)用于PCR扩增S25核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：51所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：52所示；
(26)用于PCR扩增S26核苷酸序列片段的引物对，其上游引物的核苷酸序列如序 列表中SEQ ID NO：53所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：54所示；
(27)用于PCR扩增S27核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：55所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：56所示；
(28)用于PCR扩增S28核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：57所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：58所示；
(29)用于PCR扩增S29核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：59所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：60所示；
(30)用于PCR扩增S30核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：61所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：62所示。
在一些实施方式中，本发明提供的用于PCR扩增牛结节性皮肤病病毒的全基因组序列的引物组合可以用于任何已知牛结节性皮肤病病毒，或者本发明涉及的牛结节皮肤病病毒LSDV/CH/JY/2021。
本发明又一方面还提供一种获得牛结节性皮肤病病毒的全基因组序列的方法，其包括以下步骤：
1)提取牛结节皮肤病病毒的总DNA；
2)以DNA为模板，在权利要求5提及的引物组合的引导下，用PCR方法扩增牛结节皮肤病病毒全基因组的30个核苷酸序列片段：S1(485bp)、S2(1471bp)、S3(432bp)、S4(9274bp)、S5(7408bp)、S6(5941bp)、S7(13325bp)、S8(6727bp)、S9(5492bp)、S10(947bp)、S11(9845bp)、S12(8650bp)、S13(6747bp)、S14(445bp)、S15(5291bp)、S16(1255bp)、S17(9647bp)、S18(145bp)、S19(7826bp)、S20(4292bp)、S21(8402bp)、S22(10044bp)、S23(7788bp)、S24(448bp)、S25(9712bp)、S26(9093bp)、S27(8711bp)、S28(432bp)、S29(1471bp)、S30(426bp)；
3)将30个目的基因片段回收、测序；
4)依次将步骤3)中得到的30个核苷酸序列片段的DNA序列重叠部分进行拼接、编辑及校正，得到第一初始全基因组序列；
5)将步骤1)提取得到的牛结节皮肤病病毒的总DNA进行高通量测序，并将得到的高通量测序原始数据根据参考序列与接头引物进行拼接、编辑及校正，得到第二初始全基因组序列；
6)将步骤4)得到的第一初始基因组序列补全步骤5)得到的第二初始全基因组序列，得到牛结节皮肤病病毒的全基因组序列。
在一些实施方式中，本发明提供的获得牛结节性皮肤病病毒的全基因组序列的方法可以针对任何已知牛结节性皮肤病病毒，或者本发明涉及的牛结节皮肤病病毒LSDV/CH/JY/2021。
在一些实施方式中，步骤5)中所述参考序列为由以下GenBank登录号表示的序列中的任一条或几条：NC_003027.1、MN072619.1、KX683219.1、MW631933.1、AF325528.1、AF409137.1、MW656253.1、KX894508.1、MN995838.1、MH893760.2、MW699032.1、MN642592.1、MT130502.2、KY829023.3、MT643825.1、KY702007.1、MT992618.1、MT134042.1、MH646674.1、MW435866.1、MK441838.1、KX764645.1、AF409138.1、KX764643.1、KX764644.1、MG972412.1、MW656252.1、MN636842.1、MN636840.1、MN636838.1、MN636841.1、MN636843.1、MN636839.1、AH010683.2、MW732649.1、MZ577075.1、MZ577073.1、MZ577076.1、MZ577074.1和MW355944.1。
在一些实施方式中，步骤5)中所述接头引物的核苷酸序列如序列表中SEQ ID NO：63和SEQ ID NO：64所示。
在一些实施方式中，步骤2)中所述PCR方法的反应体系(50μL)包括：总DNA 1-2μL、KOD One TM PCR Master Mix 20-30μL、上游引物(10μM)1-2μL、下游引物(10μM)1-2μL、无核酸水(Nuclease-free Water)15-25μL。
在一些实施方式中，步骤2)中所述PCR方法的反应程序包括：95℃预变性2min；95℃变性10s、50-60℃退火30s、68℃延伸30s-360s，共35个循环；最后68℃延伸10min。
基于以上技术方案提供了牛结节皮肤病病毒LSDV/CH/JY/2021全基因组序列及其扩增引物。本发明提供的用于PCR扩增牛结节皮肤病病毒的全基因组序列的引物由30对引物组成，利用这些引物对可以扩增出牛结节皮肤病病毒(可包括LSDV/CH/JY/2021)的30个核苷酸序列片段(S1、S2、S3、S4、S5、S6、S7、S8、S9、S10、S11、S12、S13、S14、S15、S16、S17、S18、S19、S20、S21、S22、S23、S24、S25、S26、S27、S28、S29和S30)，将扩增获得的30个核苷酸序列片段的DNA序列重叠部分进行拼接、编辑及校正后，用其补全利用二代测序技术(高通量测序技术)获得的并据根据参考序列与接头引物进行拼接、编辑及校正得到的牛结节性皮肤病病毒序列，可以准确得到牛结节性皮肤病病毒(可包括LSDV/CH/JY/2021)的全基因组序列。本发明提供的牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列有利于牛结节性皮肤病病毒LSDV/CH/JY/2021的致病机制、分子流行病学、遗传学等的进一步研究，从而可以对牛结节性皮肤病病毒LSDV/CH/JY/2021的诊断试剂开发、疫苗研制等奠定重要的数据支持和理论基础。
附图说明
图1为牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组的30个核苷酸序列片段(S1、S2、S3、S4、S5、S6、S7、S8、S9、S10、S11、S12、S13、S14、S15、S16、S17、S18、S19、S20、S21、S22、S23、S24、S25、S26、S27、S28、S29和S30)的PCR扩增产物的1.0％琼脂糖凝胶电泳图谱；其中A幅示出了S1、S2、S4、S5、S6、S8、S9、S11、S12、S13、S15、S17、S19、S21、S23、S25和S29的PCR扩增产物的1.0％琼脂糖凝胶电泳图谱，B幅示出了S3、S14、S20、S24、S28和S30的PCR扩增产物的1.0％琼脂糖凝胶电泳图谱，C幅示出了S10和S16的PCR扩增产物的1.0％琼脂糖凝胶电泳图谱，D幅示出了S18的PCR扩增产物的1.0％琼脂糖凝胶电泳图谱，E幅示出了S7、S17、S21和S22的PCR扩增产物的1.0％琼脂糖凝胶电泳图谱，F幅示出了S26、S27和S30的PCR扩增产物的1.0％琼脂糖凝胶电泳图谱。
具体实施方式
以下结合具体实施例，对本发明进一步阐述。应当理解的是，具体实施例仅用于进一步说明本发明，而不是用于限制本发明的内容。
下述实施例中所用方法如无特别说明均为常规方法。具体步骤可参见：《分子克隆实验指南》(《Molecular Cloning:A Laboratory Manual》Sambrook，J.，Russell，David W.，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
实施例中涉及的序列均由生工生物工程(上海)股份有限公司合成。
实施例1：设计用于PCR扩增牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组30个核苷酸序列片段的引物
根据NCBI中牛结节性皮肤病病毒参考毒株的全基因组核苷酸序列，如GenBank：MN072619.1、KX683219.1、MW631933.1、AF325528.1、AF409137.1、MW656253.1、KX894508.1、MN995838.1、MH893760.2、MW699032.1、MN642592.1、MT130502.2、KY829023.3、MT643825.1、KY702007.1、MT992618.1、MT134042.1、MH646674.1、MW435866.1、MK441838.1、KX764645.1、AF409138.1、KX764643.1、KX764644.1、MG972412.1、MW656252.1、MN636842.1、MN636840.1、MN636838.1、MN636841.1、MN636843.1、MN636839.1、AH010683.2、MW732649.1、MZ577075.1、MZ577073.1、 MZ577076.1、MZ577074.1和MW355944.1，应用生物信息学DNAStar软件进行分析、比对、筛选和优化，最终确定将牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列分成30个核苷酸序列片段：S1(485bp)、S2(1471bp)、S3(432bp)、S4(9274bp)、S5(7408bp)、S6(5941bp)、S7(13325bp)、S8(6727bp)、S9(5492bp)、S10(947bp)、S11(9845bp)、S12(8650bp)、S13(6747bp)、S14(445bp)、S15(5291bp)、S16(1255bp)、S17(9647bp)、S18(145bp)、S19(7826bp)、S20(4292bp)、S21(8402bp)、S22(10044bp)、S23(7788bp)、S24(448bp)、S25(9712bp)、S26(9093bp)、S27(8711bp)、S28(432bp)、S29(1471bp)、S30(426bp)，分别在保守序列区域内设计PCR扩增30个核苷酸序列片段的引物，经分析、比对、筛选和优化，最终确定的引物序列如下表1所示。
表1：用于PCR扩增牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列的引物
Figure PCTCN2022085327-appb-000001
Figure PCTCN2022085327-appb-000002
Figure PCTCN2022085327-appb-000003
实施例2：获得牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列
该实施例基于实施例1得到的引物对牛结节皮肤病病毒LSDV/CH/JY/2021的总DNA进行PCR扩增，最终得到牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列，具体包括以下步骤：
1)提取牛结节皮肤病病毒LSDV/CH/JY/2021的总DNA
牛结节皮肤病病毒LSDV/CH/JY/2021的总DNA的提取按照AxyprepTM Body Fluid Viral DNA/RNA Miniprep Kit(购自AXYGEN公司)说明书操作，具体步骤如下：
(1)试剂准备：预先配制含1％(V/V)冰乙酸的异丙醇；分别在试剂Buffer W1A和Buffer W2中添加指定体积的无水乙醇。即，24mL Buffer W1A中加入17mL无水乙醇；24mL Buffer W2中加入56mL无水乙醇。
(2)取1mL毒液样品(牛结节性皮肤病病毒LSDV/CH/JY/2021在牛肾细胞(MDBK)上培养得到的毒液，测定其病毒半数组织细胞感染量(TCID 50)大于10 5.5TCID 50/mL；该毒液的获得方式为：将牛结节皮肤病病毒LSDV/CH/JY/2021接毒MDBK细胞扩大培养，细胞转瓶培养约12天左右至70％面积脱落呈现网状时收毒)加入1.5mL离心管中。反复冻融三次，10000×g离心15min，收集上清；22000×g，4℃离心2h弃上清，收集沉淀。
(3)向沉淀中加入200μL Buffer V-L，涡旋振荡混合均匀，静置5min。
(4)加入75μL Buffer V-L，涡旋振荡混合均匀，12000rpm离心5min。
(5)将上清转移至新的2mL离心管(试剂盒内提供)中，加入300μL异丙醇(1％ 冰乙酸)，上下倒置6-8次，混合均匀。
(6)将制备管置于2mL离心管(试剂盒内提供)中，取步骤(5)中的混合液移入制备管中，6000rpm离心1min。
(7)弃滤液，将制备管置回到2mL离心管中，加500μL Buffer W1A，室温静置1min，12000rpm离心1min。
(8)弃滤液，将制备管置回到2mL离心管中，加800μL Buffer W2，12000rpm离心1min。
(9)弃滤液，将制备管置回到2mL离心管中，12000rpm离心1min。
(10)将制备管置于洁净的1.5mL离心管(试剂盒内提供)中，在制备管膜中央加40μL无酶水，室温静置1min，12000×g离心1min洗脱DNA，-20℃冻存备用。
2)PCR扩增牛结节皮肤病病毒LSDV/CH/JY/2021全基因组的30个核苷酸序列片段
将步骤1)提取的牛结节性皮肤病病毒LSDV/CH/JY/2021的总DNA作为模板，在实施例1提供的引物(表1所示)的引导下，分别用PCR方法扩增牛结节性皮肤病病毒LSDV/CH/JY/2021全基因组的30个核苷酸序列片段：S1(485bp)、S2(1471bp)、S3(432bp)、S4(9274bp)、S5(7408bp)、S6(5941bp)、S7(13325bp)、S8(6727bp)、S9(5492bp)、S10(947bp)、S11(9845bp)、S12(8650bp)、S13(6747bp)、S14(445bp)、S15(5291bp)、S16(1255bp)、S17(9647bp)、S18(145bp)、S19(7826bp)、S20(4292bp)、S21(8402bp)、S22(10044bp)、S23(7788bp)、S24(448bp)、S25(9712bp)、S26(9093bp)、S27(8711bp)、S28(432bp)、S29(1471bp)、S30(426bp)，具体步骤如下：
(1)按照KOD One TM PCR Master Mix试剂盒(购自TOYOBO公司)说明书中的试剂比例配制反应体系，具体组分如下表2所示。
表2：PCR扩增牛结节性皮肤病病毒LSDV/CH/JY/2021全基因组的30个核苷酸序列片段的反应体系
Figure PCTCN2022085327-appb-000004
(2)将按照上表2配置好的试剂放入PCR仪中进行PCR扩增，PCR反应程序如下表3所示。
表3：PCR扩增牛结节性皮肤病病毒LSDV/CH/JY/2021全基因组的30个核苷酸序列片段的反应程序
Figure PCTCN2022085327-appb-000005
注：表3中，短片段指长度为2000bp以下的片段，长片段指长度为2000bp以上的片段。
3)PCR扩增片段的回收、连接、转化和测序
将3’与5’端的目的核苷酸序列片段(S1和S30)回收、连接、转化，挑取阳性单克隆菌，送生工生物工程(上海)股份有限公司测序，剩余的28个目的核苷酸片段回收后直接送生工生物工程(上海)股份有限公司进行测序。具体包括以下步骤：
(1)将步骤2)PCR扩增的牛结节皮肤病病毒LSDV/CH/JY/2021全基因组的30个核苷酸序列片段进行1.0％琼脂糖凝胶电泳检测，检测结果如图1所示(泳道S1(485bp)、泳道S2(1471bp)、泳道S3(432bp)、泳道S4(9274bp)、泳道S5(7408bp)、泳道S6(5941bp)、泳道S7(13325bp)、泳道S8(6727bp)、泳道S9(5492bp)、泳道S10(947bp)、泳道S11(9845bp)、泳道S12(8650bp)、泳道S13(6747bp)、泳道S14(445bp)、泳道S15(5291bp)、泳道S16(1255bp)、泳道S17(9647bp)、泳道S18(145bp)、泳道S19(7826bp)、泳道S20(4292bp)、泳道S21(8402bp)、泳道S22(10044bp)、泳道S23(7788bp)、泳道S24(448bp)、泳道S25(9712bp)、泳道S26(9093bp)、泳道S27(8711bp)、泳道S28(432bp)、泳道S29(1471bp)、泳道S30(426bp))，根据图1所示的结果可见，PCR扩增的30个目的核苷酸序列片段与预期相符。按照Thermo Scientific GeneJET Gel Extraction Kit试剂盒(购自ThermoFisher公司)说明书，回收并纯化30个目的核苷酸序列片段，具体步骤如下：
(1.1)试剂准备：向Wash Buffer中添加指定体积的无水乙醇(具体为9mL中加入45mL无水乙醇)。
(1.2)在紫外灯下，用干净的刀片切下含有目的基因片段的琼脂糖凝胶块，将其放入1.5mL灭菌离心管中(先称空的1.5mL离心管重量，然后再称放入琼脂糖凝胶块的1.5mL离心管重量)。
(1.3)计算琼脂糖凝胶块重量，按1mg＝1μL计算琼脂糖凝胶块的体积。
(1.4)向盛有胶块的1.5mL离心管中，加入1:1凝胶体积量(琼脂糖凝胶浓度为1.0％)的Binding Buffer。
(1.5)均匀混合后，50℃-60℃溶解胶块，此时应间断振荡混合，使胶块充分溶解(约5-10分钟)。
(1.6)将试剂盒中的GeneJET Purification Column安置于收集管上。
(1.7)GeneJET Purification Column中加入100Binding Buffer室温静置1min，10000rpm离心1min，弃滤液，后将步骤(1.5)的溶液转移至GeneJET Purification Column中，12000rpm离心1min，弃滤液。
(1.8)将700μL的Wash Buffer加入GeneJET Purification Column中，12000rpm离心1min，弃滤液。
(1.9)重复步骤(1.8)。
(1.10)将GeneJET Purification Column安置于收集管上，12000rpm离心1min。
(1.11)将GeneJET Purification Column安置于新的1.5mL的离心管上，在GeneJET Purification Column膜中央处加入30μL灭菌水或Elution Buffer，室温静置1min。
(1.12)12000rpm离心1min洗脱DNA。
(2)连接目的基因片段
(2.1)将上述步骤(1)回收纯化的3’与5’目的核苷酸序列片段(S1和S30)分别连接到
Figure PCTCN2022085327-appb-000006
载体(购自Life technologies invitrogen公司)中，连接时按照
Figure PCTCN2022085327-appb-000007
载体说明书中的试剂比例配制反应体系，反应体系如下表4所示。
表4：用
Figure PCTCN2022085327-appb-000008
载体连接3’与5’目的核苷酸序列片段的反应体系
Figure PCTCN2022085327-appb-000009
(2.2)将上表4中的各种试剂加入0.2mL离心管中，轻轻混匀，室温连接5-15min。反应结束后将PCR管置于冰上冷却。
(3)转化：将3’与5’目的核苷酸序列片段(S1和S30)的连接产物分别转化到E.Coli JM109感受态细胞(购自TaKaRa公司)中，具体步骤如下：
(3.1)将E.Coli JM109感受态细胞从-80℃冰箱取出，置于冰上融化。
(3.2)将6μL连接产物移至100μL E.Coli JM109感受态细胞中，混匀，冰浴30min。
(3.3)42℃热激45s，迅速置于冰上2-5min。
(3.4)加入800μL SOC液体培养基(购自TaKaRa公司)，37℃、150rpm振荡培养1h，使细菌复苏并表达质粒编码的抗生素抗性基因。
(3.5)取200μL步骤(3.4)中的菌液，转移到含50μg/mL氨苄青霉素的LB琼脂平板上，均匀涂板，待全部菌液被吸收后，37℃温箱倒置培养12-14h。
(4)筛选阳性克隆
挑取上述步骤(3)中的LB琼脂平板上长出的白色单一菌落，分别接种于3mL LB液体培养基中(含50μg/mL氨苄青霉素)，37℃、200rpm振摇过夜(12-16h)。选取阳性单克隆菌，送生工生物工程(上海)股份有限公司进行测序。剩余的28个目的核苷酸片段回收后直接送生工生物工程(上海)股份有限公司进行测序。
4)将上述步骤1)中提取到的牛结节皮肤病病毒LSDV/CH/JY/2021的总DNA送生工生物工程(上海)股份有限公司建文库进行高通量测序(即二代测序)。
5)用生物信息学软件依次将上述步骤3)中测序得到的30个核苷酸序列片段：S1(485bp)、S2(1471bp)、S3(432bp)、S4(9274bp)、S5(7408bp)、S6(5941bp)、S7(13325bp)、S8(6727bp)、S9(5492bp)、S10(947bp)、S11(9845bp)、S12(8650bp)、S13(6747bp)、S14(445bp)、S15(5291bp)、S16(1255bp)、S17(9647bp)、S18(145bp)、S19(7826bp)、S20(4292bp)、S21(8402bp)、S22(10044bp)、S23(7788bp)、S24(448bp)、S25(9712bp)、S26(9093bp)、S27(8711bp)、S28(432bp)、S29(1471bp)、S30(426bp)的DNA序列重叠部分进行拼接、信息编辑及校正分析，得到第一初始全基因组序列；再将步骤4)中得到的二代测序(即高通量测序)原始数据(若干基因片段)根据参考序列(该实施例使用的参考序列具体为Lumpy skin disease virus NI-2490,complete genome NC_003027.1)与接头引物(作为标签引物，其上下游的核苷酸序列分别如序列表中SEQ ID NO：63和SEQ ID NO：64所示)进行拼接、信息编辑及校正分析，得到第二初始全基因组序列。利用第一初始全基因组序列补全第二初始全基因组序列(由二代测序技术获得的第二初始全 基因组序列可能是不完整的，缺少某些小的基因片段，尤其是全基因组序列的3’端和5’端序列，因此需要使用由30对引物扩增获得的第一初始全基因组序列对第二初始全基因组序列进行补全)，补全后的第二初始全基因组序列作为牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列，其核苷酸序列如序列表中SEQ ID NO：1所示。
对SEQ ID NO：1所示的DNA序列进行分析可知，牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列由150606个核苷酸组成，自5’端第336bp-2527bp(2192bp)与自5’端第148415bp-150606bp(2192bp)互为回文结构倒置重复；自5’端第9bp-371bp开始为第1个编码(CDS)序列，自5’端第150120bp-150599bp为第157个编码(CDS)序列，即根据全基因组序列预测的牛结节性皮肤病病毒LSDV/CH/JY/2021的编码(CDS)序列的核苷酸(DNA)序列为SEQ ID NO：1中自5’端第9bp-150599bp所示，长150591bp，另外也可利用牛结节性皮肤病病毒LSDV/CH/JY/2021的全基因组序列参考现有数据库(例如NR、SwissProt、Pfam、COG_category、GO、KEGG_pathway、PHI和TCDB数据库)比对出牛结节性皮肤病病毒LSDV/CH/JY/2021的编码序列(CDS)图谱。经分析并参考现有的牛结节性皮肤病病毒的蛋白序列，可知牛结节性皮肤病病毒LSDV/CH/JY/2021的编码序列(CDS)编码的蛋白序列如序列表中SEQ ID NO:2所示，序列表中的SEQ ID NO:2由157个编码序列(CDS)编码获得。
将牛结节性皮肤病病毒LSDV/CH/JY/2021与NCBI(美国国家生物技术信息中心)中山羊痘病毒属(牛结节性皮肤病病毒种、绵羊痘病毒种和山羊痘病毒种)70株参考毒株(MN072624.1、MN072625.1、MN072623.1、MN072621.1、KC951854.1、MH381810.1、MN072620.1、MW020570.1、MN072622.1、AY077836.1、KX576657.1、AY077835.1、MN072628.1、MN072629.1、AY077832.1、MW167071.1、MT137384.1、AY077833.1、MN072631.1、MN072630.1、MW167070.1、MW020571.1、AY077834.1、KT438551.1、KT438550.1、MG000157.1、MN072627.1、MN072626.1、MG000156.1、AF124517.1、MN072619.1、AF325528.1KX683219.1、MW631933.1、AF409137.1、MW656253.1、KY702007.1、KX894508.1、MN995838.1、MH893760.2、MW699032.1、MN642592.1、MT130502.2、KY829023.3、MT643825.1、MT992618.1、MT134042.1、MH646674.1、AH010683.2、MW435866.1、MW656252.1、MK441838.1、KX764645.1、AF409138.1、MG972412.1、KX764643.1、KX764644.1、MN636839.1、MN636843.1、MN636841.1、MN636838.1、MN636842.1、MN636840.1、MW732649.1、MZ577075.1、MZ577073.1、MZ577076.1、MZ577074.1、MW355944.1、MK860688.1)的全基因组序列制作遗传进化树，结果表明牛结节性皮肤病病毒LSDV/CH/JY/2021与LSDV病毒种的参考毒株位于同一分支内，表明LSDV/CH/JY/2021属于LSDV病毒种，且证明LSDV/CH/JY/2021同 China/GD01/2020(MW355944.1)同源性最高。
工业应用性
本发明提供了牛结节皮肤病病毒LSDV/CH/JY/2021的全基因组序列及其扩增引物，该牛结节皮肤病病毒LSDV/CH/JY/2021的全基因组序列可准确反映牛结节皮肤病病毒LSDV/CH/JY/2021的基因组信息，利用其有利于该病毒的致病机制等的研究，并有利于针对该病毒的诊断试剂开发和疫苗研制等。

Claims (10)

  1. 一种牛结节皮肤病病毒的全基因组序列，该牛结节皮肤病病毒命名为LSDV/CH/JY/2021，所述全基因组序列的核苷酸序列如序列表中SEQ ID NO:1所示，或在高严谨条件下可与序列表中SEQ ID NO:1所示的核苷酸序列杂交的核苷酸序列。
  2. 根据权利要求1所述的牛结节皮肤病病毒的全基因组序列，其中在SEQ ID NO:1所示的核苷酸序列中，自5’端第336bp-2527bp与自5’端第148415bp-150606bp互为回文结构倒置重复，自5’端第9bp-150599bp为编码(CDS)序列。
  3. 权利要求1或2所述的牛结节皮肤病病毒的全基因组序列及其编码(CDS)序列编码的蛋白序列。
  4. 根据权利要求3所述的蛋白序列，其中所述蛋白序列的氨基酸序列如序列表中SEQ ID NO：2所示。
  5. 用于PCR扩增牛结节性皮肤病病毒的全基因组序列的引物组合，其由30对引物组成，分别用于扩增牛结节性皮肤病病毒的全基因组的30个核苷酸序列片段：S1、S2、S3、S4、S5、S6、S7、S8、S9、S10、S11、S12、S13、S14、S15、S16、S17、S18、S19、S20、S21、S22、S23、S24、S25、S26、S27、S28、S29和S30，所述引物组合包括：
    (1)用于PCR扩增S1核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：3所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：4所示；
    (2)用于PCR扩增S2核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：5所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：6所示；
    (3)用于PCR扩增S3核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：7所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：8所示；
    (4)用于PCR扩增S4核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：9所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：10所示；
    (5)用于PCR扩增S5核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：11所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：12所示；
    (6)用于PCR扩增S6核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：13所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：14所示；
    (7)用于PCR扩增S7核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：15所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：16所示；
    (8)用于PCR扩增S8核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：17所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：18所示；
    (9)用于PCR扩增S9核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：19所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：20所示；
    (10)用于PCR扩增S10核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：21所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：22所示；
    (11)用于PCR扩增S11核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：23所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：24所示；
    (12)用于PCR扩增S12核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：25所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：26所示；
    (13)用于PCR扩增S13核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：27所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：28所示；
    (14)用于PCR扩增S14核苷酸序列片段的引物对，其上游引物的核苷酸序列如 序列表中SEQ ID NO：29所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：30所示；
    (15)用于PCR扩增S15核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：31所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：32所示；
    (16)用于PCR扩增S16核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：33所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：34所示；
    (17)用于PCR扩增S17核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：35所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：36所示；
    (18)用于PCR扩增S18核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：37所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：38所示；
    (19)用于PCR扩增S19核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：39所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：40所示；
    (20)用于PCR扩增S20核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：41所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：42所示；
    (21)用于PCR扩增S21核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：43所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：44所示；
    (22)用于PCR扩增S22核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：45所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：46所示；
    (23)用于PCR扩增S23核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：47所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：48所示；
    (24)用于PCR扩增S24核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：49所示，其下游引物的核苷酸序列如序列表中SEQ ID NO： 50所示；
    (25)用于PCR扩增S25核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：51所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：52所示；
    (26)用于PCR扩增S26核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：53所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：54所示；
    (27)用于PCR扩增S27核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：55所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：56所示；
    (28)用于PCR扩增S28核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：57所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：58所示；
    (29)用于PCR扩增S29核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：59所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：60所示；
    (30)用于PCR扩增S30核苷酸序列片段的引物对，其上游引物的核苷酸序列如序列表中SEQ ID NO：61所示，其下游引物的核苷酸序列如序列表中SEQ ID NO：62所示。
  6. 一种获得牛结节性皮肤病病毒的全基因组序列的方法，其包括以下步骤：
    1)提取牛结节皮肤病病毒的总DNA；
    2)以DNA为模板，在权利要求5提及的引物组合的引导下，用PCR方法扩增牛结节皮肤病病毒全基因组的30个核苷酸序列片段：S1(485bp)、S2(1471bp)、S3(432bp)、S4(9274bp)、S5(7408bp)、S6(5941bp)、S7(13325bp)、S8(6727bp)、S9(5492bp)、S10(947bp)、S11(9845bp)、S12(8650bp)、S13(6747bp)、S14(445bp)、S15(5291bp)、S16(1255bp)、S17(9647bp)、S18(145bp)、S19(7826bp)、S20(4292bp)、S21(8402bp)、S22(10044bp)、S23(7788bp)、S24(448bp)、S25(9712bp)、S26(9093bp)、S27(8711bp)、S28(432bp)、S29(1471bp)、S30(426bp)；
    3)将30个目的基因片段回收、测序；
    4)依次将步骤3)中得到的30个核苷酸序列片段的DNA序列重叠部分进行拼接、编辑及校正，得到第一初始全基因组序列；
    5)将步骤1)提取得到的牛结节皮肤病病毒的总DNA进行高通量测序，并将得到的高通量测序原始数据根据参考序列与接头引物进行拼接、编辑及校正，得到第二初始全基因组序列；
    6)将步骤4)得到的第一初始基因组序列补全步骤5)得到的第二初始全基因组序列，得到牛结节皮肤病病毒的全基因组序列。
  7. 根据权利要求6所述的方法，步骤5)中所述参考序列为由以下GenBank登录号表示的序列中的任一条或几条：NC_003027.1、MN072619.1、KX683219.1、MW631933.1、AF325528.1、AF409137.1、MW656253.1、KX894508.1、MN995838.1、MH893760.2、MW699032.1、MN642592.1、MT130502.2、KY829023.3、MT643825.1、KY702007.1、MT992618.1、MT134042.1、MH646674.1、MW435866.1、MK441838.1、KX764645.1、AF409138.1、KX764643.1、KX764644.1、MG972412.1、MW656252.1、MN636842.1、MN636840.1、MN636838.1、MN636841.1、MN636843.1、MN636839.1、AH010683.2、MW732649.1、MZ577075.1、MZ577073.1、MZ577076.1、MZ577074.1和MW355944.1。
  8. 根据权利要求6或7所述的方法，步骤5)中所述接头引物的核苷酸序列如序列表中SEQ ID NO：63和SEQ ID NO：64所示。
  9. 根据权利要求6-8中任一项所述的方法，步骤2)中所述PCR方法的反应体系(50μL)包括：总DNA 1-2μL、KOD One TMPCR Master Mix 20-30μL、上游引物(10μM)1-2μL、下游引物(10μM)1-2μL、无核酸水(Nuclease-free Water)15-25μL。
  10. 根据权利要求6-9中任一项所述的方法，步骤2)中所述PCR方法的反应程序包括：95℃预变性2min；95℃变性10s、50-60℃退火30s、68℃延伸30s-360s，共35个循环；最后68℃延伸10min。
PCT/CN2022/085327 2022-04-06 2022-04-06 牛结节性皮肤病病毒lsdv/ch/jy/2021的全基因组序列及其扩增引物 WO2023193140A1 (zh)

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AU2021102363A4 (en) * 2020-09-29 2021-06-24 Institute Of Animal Health, Guangdong Academy Of Agricultural Sciences Dual-fluorescent pcr primer, probe, method and kit for identifying capripox virus and lumpy skin disease virus

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AU2021102363A4 (en) * 2020-09-29 2021-06-24 Institute Of Animal Health, Guangdong Academy Of Agricultural Sciences Dual-fluorescent pcr primer, probe, method and kit for identifying capripox virus and lumpy skin disease virus

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