Application of Bovine SP110 Gene Splicing Editing in Enhancing Bovine Anti-Tuberculosis Ability

Abstract

The present invention discloses the application of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle, belonging to the technical field of bovine anti-tuberculosis breeding. The present invention starts from splicing-related cis-acting elements, and regulates the alternative splicing reaction of bovine SP110 by introducing cis-acting elements, thereby affecting the anti-tuberculosis ability of cattle. The cis-acting element sequence is named the pre-SAND-exon sequence, which is located in exon 12 and the adjacent 5'-terminal intron 12 of the human or equine SP110 gene, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The present invention first regulates the alternative splicing of the bovine SP110 gene by introducing the pre-SAND-exon sequence into the bovine SP110 gene, reduces the expression abundance of the spliceosome bSP110c with lower anti-tuberculosis ability, and regulates the higher abundances of the spliceosomes bSP110a and bSP110b with anti-tuberculosis infection ability. The provided bovine SP110 gene splicing editing can effectively improve the anti-tuberculosis ability of cattle, can be used for the breeding of anti-tuberculosis cattle, effectively simplifies the breeding method, improves the breeding efficiency, and saves the breeding cost.

Description

Technical Field

[0001] The present invention relates to the technical field of bovine tuberculosis resistance breeding, and in particular to the application of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of bovines. Background Art

[0002] Bovine tuberculosis (BTB) is a chronic, debilitating zoonosis primarily caused by infection with Mycobacterium bovis (M. bovis). According to statistics, approximately 50 million cattle worldwide are TB-positive annually, resulting in approximately $3 billion in agricultural losses. Furthermore, approximately 10%-15% of human pulmonary tuberculosis infections are caused by bovine tuberculosis, threatening animal-source food safety and public health. In recent years, my country's cattle industry has continued to expand. While ensuring the supply of animal products, dairy bovine tuberculosis has become a key issue affecting the sustainable development of the industry. Summary of the Invention

[0003] The purpose of the present invention is to provide an application of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle, so as to provide a sequence closely linked to the anti-tuberculosis ability of cattle for the effective prevention and control of bovine tuberculosis and the breeding of anti-tuberculosis cattle breeds.

[0004] To achieve the above objectives, the present invention provides the use of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle. The splicing editing is achieved by introducing a cis-acting element. The introduced cis-acting element is the 12th exon and the adjacent 5′-end 12th intron partial sequence of the human or horse SP110 gene. The sequence derived from humans is shown in SEQ ID NO.1, and the sequence derived from horses is shown in SEQ ID NO.2.

[0005] Preferably, the application is to regulate the variable splicing of bovine SP110 by introducing cis-acting elements, thereby improving the anti-tuberculosis ability of cattle.

[0006] Preferably, the application is achieved by regulating the alternative splicing of bovine SP110 by introducing a cis-acting element upstream of the SP110 gene, thereby reducing the abundance of the transcript bSP110c and increasing the abundance of bSP110a and bSP110b transcripts. The amino acid sequence of the bSP110c transcript is shown in SEQ ID NO.8, the amino acid sequence of the bSP110a transcript is shown in SEQ ID NO.6, and the amino acid sequence of the bSP110b transcript is shown in SEQ ID NO.7.

[0007] The application of bovine SP110 gene splicing editing in promoting the expression of inflammatory factors as described above is achieved by regulating bovine SP110 alternative splicing by introducing a cis-acting element upstream of the SP110 gene, thereby reducing the abundance of transcript bSP110c and increasing the abundance of bSP110a and bSP110b.

[0008] The use of the bovine SP110 gene splicing editing as described above, and the vector or engineered bacteria containing the bovine SP110 gene splicing editing in reducing the intracellular proliferation ability of Mycobacterium tuberculosis.

[0009] The use of the bovine SP110 gene splicing and editing as described above, and the vector or engineered bacteria containing the bovine SP110 gene splicing and editing in the preparation of anti-tuberculosis preparations.

[0010] The application of the bovine SP110 gene splicing editing as described above, and the vector or engineered bacteria containing the bovine SP110 gene splicing editing in the breeding of tuberculosis-resistant dairy cows.

[0011] The present invention introduces a cis-acting element (pre-SAND-exon sequence) into the bovine SP110 gene, which can significantly reduce the abundance of bovine bSP110c transcripts and increase the abundance of bSP110a and bSP110b transcripts, thereby increasing the expression of inflammatory factors during the process of Mycobacterium tuberculosis infection of macrophages, reducing the proliferation ability of Mycobacterium tuberculosis in cells, and improving anti-tuberculosis ability.

[0012] Therefore, the application of bovine SP110 gene splicing editing provided by the present invention in improving the anti-tuberculosis ability of cattle has the following specific technical effects:

[0013] (1) The present invention regulates the splicing and editing of the bovine SP110 gene by introducing the 12th exon of the human or horse SP110 gene and the adjacent 5′-end 12th intron partial sequence (pre-SAND-exon sequence) into the bovine SP110 gene, thereby effectively improving the anti-tuberculosis ability of cattle;

[0014] (2) The splicing editing of the bovine SP110 gene provided by the present invention can be used for breeding tuberculosis-resistant cattle, providing new materials for breeding disease-resistant dairy cows.

[0015] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0017] Figure 1 This is a gel electrophoresis diagram of the enzyme digestion product in Example 1 of the present invention;

[0018] Figure 2 is a schematic diagram of the functional domains of the three bSP110 transcripts drawn in Example 2 of the present invention;

[0019] Figure 3 This is the CFU analysis result in Example 4 of the present invention;

[0020] Figure 4 This is the ELISA test result in Example 4 of the present invention;

[0021] Figure 5 Schematic diagram of the pCMV-Flag-ASVV vector constructed in Example 5 of the present invention;

[0022] Figure 6 Schematic diagram of the pCMV-Flag-Rhuman / Rhorse-ASVV vector constructed in Example 6 of the present invention;

[0023] Figure 7 Schematic diagram of the Replace Knock-in human / horse SP110 element Minigene vector constructed in Example 7 of the present invention;

[0024] Figure 8 is a position map of the quantitative primers in Example 8 of the present invention;

[0025] Figure 9 The mRNA levels of the three transcripts of the bovine SP110 gene expressed by the three minigene reporter vectors constructed in Examples 5, 6 and 7 of Example 8 of the present invention;

[0026] Figure 10 This is the CFU analysis result in Example 9 of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0028] In order to make the purpose, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments, which is intended to provide further details of the present invention. Unless otherwise indicated, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs, and experimental methods not particularly described are conventional experimental methods in this area.

[0029] The bovine SP110 gene splicing editing provided by the present invention can reduce the production of a transcript bSP110c (amino acid sequence as shown in SEQ ID NO.9) of the bovine SP110 gene that has no anti-tuberculosis function by regulating the variable splicing of the bovine SP110 gene, while increasing the abundance of spliceosomes of SP110a (amino acid sequence as shown in SEQ ID NO.7) and SP110b (amino acid sequence as shown in SEQ ID NO.8) with anti-tuberculosis infection ability, thereby improving the anti-tuberculosis ability of cattle.

[0030] SEQ ID NO.7:

[0031] MTRALEKALLQHFIQQKLEIAYAINKPFPFFEGLRDNFFITERLYRESMEACENLVPLSRVVYNILTQLEKTFSLSFLKILFSQINLNEYPNLITTLNSFTRVVTSHGGWSRITTTLLEAPANPAGRSSPRTLLLPRLPCRQPLRSLPPCGPSVSERAPAQRSSEVLRELPSRTGLTMTLRETIQKGRLSPVSSDNLTPQIKDKEDTQEMPCTSSDPVPVKRDDVPEPSDPKELQEASRTLPSKKGKKRKRCIWATSKKRQQKNSHQGSKMRKQEGAGGQRQLIPVDQGAALPGHGIQKTLQGLDQVTHTEDDSIRNTKVMTRTQKRRTECAQMPKSEEISDNTSEMDEGKRSQEPPSTPPRITHGKDSMDDDSKLSLEKSPGKKRAASSGLRIQEKLQGVNPVTQKKDDSTRKSTVMTRARKAQAEGARMPAQKDKAGGGTGHLNAQRGRVSHGARTEKPKDKTIDFHSPELPVTCGEAKGILYKEKMKQGSSKKCIQNEKGVWFTPTEFEIEGKRKHSRNWKHSVFCGGKSLGKLLQNGILFCPSRKHLKTEQENSKECAVCCKGGSLLRCDTCPRFFHEDCHIPPAEAERSPWSCTFCRMKESSGNQQGLQESEVWARSMQLEEQLKCEFLLLKAYCHPQSSFFAETPCNIRDYGEPFREAMWLDLVKERLTEKVYTMAWFLRDMRLIFHNHKTFYKASDFGQVGLDLEAEFEKDLKEVLICHEVSDNSFQAHP

[0032] SEQ ID NO.8:

[0033] MTRALEKALLQHFIQQKLEIAYAINKPFPFFEGLRDNFFITERLYRESMEACENLVPLSRVVYNILTQLEKTFSLSFLKILFSQINLNEYPNLITTLNSFTRVVTSHGGWSRITTTLLEAPANPAGRSSPRTLLPRLPCRQPLRSLPPCGPSVSERAPAQRSSEVLRELPSRTGLTMTLRETIQKGRLSPVSSDNLTPQIKDKEDTQEMPCTSDPVPVKRDDVPEPSDPKELQEASRTLPSKKGKKRKRCIWATSKKRQQKNSHQGSKMRKQEGAGG QRQLIPVDQGAALPGHGIQKTLQGLDQVTHTEDDSIRNTKVMTRTQKRRTECAQMPKSEEISDNTSEMDEGKRSQEPPSTPPRITHGKDSMDDDSKLSLEKSPGKKRAASSGLRIQEKLQGVNQVTQKKDDSTRKSTVMTRARKAQAEGARMPAQKDKAGGGTGHLNAQRGRVSHGARTEKSKDKTIDFHSPELPVTCGGAKGILYKEKMKQGSSKKCIQNEKGVWFTPTEFEIEGKRKHSRNWKHSVFCGGKSLGKLLQNGILFCPSRKHLKTEVNSR

[0034] SEQ ID NO.9:

[0035] MTRALEKALLQHFIQQKLEIAYAINKPFPFFEGLRDNFFITERLYRESMEACENLVPLSRVVYNILTQLEKTFSLSFLKILFSQINLNEYPNLITTLNSFTRVVTSHGGWSRITTT LLEAPANPAGRSSPRTLLLPRLPCRQPLRSLPPCGPSVSERAPAQRSSEVLRELPSRTGLTMTLRETIQKGRLSPVSSDNLTPQIKDKEDTQEMPCTSSDPVPVKRDDVPEPSDPK ELQEASRTLPSKKGKKRKRCIWATSKKRQQKNSHQGSKMRKQEGAGGQRQLIPVDQGAALPGHGIQKTLQGLDQVTHTEDDSIRNTKVMTRTQKRRTECAQMPKSEEISDNTSEMD EGKRSQEPPSTPPRITHGKDSMDDDSKLSLEKSPGKKRAASSGLRIQEKLQGVNQVTQKKDDSTRKSTMTRARKAQAEGARMPAQKDKAGGGTGHLNAQRGRVSHGARTGDVDLK

[0036] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0037] Example 1

[0038] The three transcripts of the bovine SP110 gene were cloned as follows:

[0039] S1.1. Four 3′ RACE primers were designed based on the bovine SP110 mRNA sequence predicted in the NCBI database, as shown in Table 1:

[0040] Table 1

[0041] Primer name Primer sequence (5′-3′) SP110-3′RACE-NGSP-1 GTCTCCCCTGCCGACAACCCCTAC(SEQ ID NO.3) SP110-3′RACE-NGSP-2 AAGGAAGGCTCAGTCCAGTGTCCAG(SEQ ID NO.4) SP110-3′RACE-NGSP-3 CCTGAACCAAGCGACCCAAAGGA(SEQ ID NO.5) SP110-3′RACE-NGSP-4 ATGATGTCCCTGAACCAAGCGACCCA(SEQ ID NO.6)

[0042] S1.2. Total RNA from bovine macrophages was extracted using the Trizol method. The first reverse transcription was performed using the reverse transcription system shown in Table 2. The reaction conditions were: 72°C for 3 min, 42°C for 2 min. The cells were then quickly placed on ice for a second reverse transcription using the system shown in Table 3. The reaction conditions were: 42°C for 90 min, 70°C for 10 min. cDNA was obtained, a portion of which was used for the next step of the experiment, and the remainder was frozen at -80°C for later use.

[0043] Table 2

[0044] Components Volume RNASample 1 μg 3'-CDSPrimerA 1 μL RandomPrimer 1 μL <![CDATA[RNaseFreeddH2O]]> To 12 μL Total volume 12 μL

[0045] Table 3

[0046] Components Volume The above 12 μL reaction solution 12 μL 5×First-StandardBuffer 4μL dNTPs 1 μL RNase Inhibitor 0.5μL DDT 0.5μL SMARTScribeReverseTranscriptase 2μL Total volume 20 μL

[0047] S1.3, using the system shown in Table 4 and the amplification conditions shown in Table 5, nested PCR was performed using the cDNA obtained in step S1.2 as a template to clone the SP110 transcript. The next nested PCR was performed using the system shown in Table 6 and the amplification conditions shown in Table 7, using the product of the first step as a template. The PCR products were separated by agarose gel electrophoresis, and the amplification results were shown in FIG. Figure 1 All the bands were cut out and purified using an agarose gel extraction kit.

[0048] Table 4

[0049] Component Volume <![CDATA[PCR-GradeH2O]]> 15.5μL 2×SeqAmpBuffer 25 μL SeqAmpDNAPolymerase 1 μL template 2.5 μL 10×UPM 5μL SP110-3'RACE-NGSP 1 μL TotalVolume 50μL

[0050] Table 5

[0051] temperature time Number of cycles 94℃ 30s 5 72℃ 2min 5 94℃ 30s 5 70℃ 30s 5 72℃ 2min 5 94℃ 30s 5 68℃ 30s 5 72℃ 2min 5 16℃ ∞ 1

[0052] Table 6

[0053]

[0054]

[0055] Table 7

[0056] temperature time Number of cycles 94℃ 30s 20 68℃ 30s 20 72℃ 2min 20

[0057] S1.4. Prepare a ligation system using the components shown in Table 8 using the recovered product obtained in step S1.3. Mix thoroughly and ligate overnight at 4°C. The ligation product is then transformed into Escherichia coli DH5α. Single colonies are screened using ampicillin and propagated in liquid LB medium by shaking. When the OD reaches 0.6, the plasmid is extracted using a plasmid extraction kit.

[0058] Table 8

[0059] Component Volume Rubber recycling products 4.5 μL pMD19T (simple) vector 0.5μL SolutionⅠ 5μL Total volume 10 μL

[0060] The plasmid was sent to the company for sequencing analysis, and three major transcripts of bovine SP110 were obtained, named bSP110a, bSP110b and bSP110c, with amino acid sequences of SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0061] Example 2

[0062] The three transcripts obtained in Example 1 were subjected to domain analysis as follows:

[0063] The three bSP110 transcripts obtained in Example 1 were translated into amino acid sequences, and then the conserved domains were predicted using the Conserved-Domain Search online tool in the NCBI database. Based on the analysis results, a schematic diagram of the functional domains of the bSP110 gene was drawn. The results are shown in FIG. Figure 2 As shown, the C-terminal functional domains of the three bSP110 transcripts bSP110a, bSP110b and bSP110c decrease in sequence, among which the transcript bSP110c lacks the core functional domain SAND.

[0064] Example 3

[0065] The eukaryotic expression vectors of the three transcripts obtained in Example 1 were constructed as follows:

[0066] S3.1. Using the frozen cDNA from step S1.2 of Example 1 as a template, one upstream primer and three downstream primers were used to clone the ORFs of the three major SP110 spliceosomes, bSP110a, bSP110b, and bSP110c, respectively. The primer sequences are shown in Table 9 (lowercase letters represent introduced restriction sites, F represents upstream primers, and R represents downstream primer sequences):

[0067] Table 9

[0068]

[0069]

[0070] S3.2. The PCR products were recovered from agarose gel and then subjected to an enzyme digestion reaction. The enzyme digestion reaction system is shown in Table 10. After reacting at 37° C. for 3 h, the products were recovered by agarose gel electrophoresis and then ligated with the pCMV-HA vector (the ligation method was the same as in step S1.4 of Example 1). The obtained recombinant expression vectors were named: pCMV-HA-bSP110a, pCMV-HA-bSP110b, and pCMV-HA-bSP110c, respectively.

[0071] Table 10

[0072] Components Volume Vector / PCRProduct 2 μg EcoRI 1 μL XOt 1 μL 10 × CutSmart 5μL ddH2O Up to 50 μL

[0073] Example 4

[0074] The anti-tuberculosis ability of the three transcripts obtained in Example 3 was investigated in the following steps:

[0075] S4.1. The eukaryotic expression vectors pCMV-HA-SP110a, pCMV-HA-SP110b, and pCMV-HA-SP110c constructed in Example 3 were used to transfect the RAW264.7 cell line. After 24 hours of expression, the cells were infected with H37Rv Mycobacterium tuberculosis. The steps were as follows:

[0076] 1) Cell transfection and infection with Mycobacterium tuberculosis

[0077] Mycobacterium tuberculosis strains H37Rv and M. bovis were obtained from the research group led by Guo Aizhen at Huazhong Agricultural University. This bacterial challenge experiment was conducted entirely in the Animal Biosafety Level 3 Laboratory at Huazhong Agricultural University.

[0078] First, RAW264.7 cells were transfected with the constructed eukaryotic expression vectors pCMV-HA-bSP110a, pCMV-HA-bSP110b, and pCMV-HA-bSP110c in 12-well plates. Cells were cultured to 70% confluency before transfection. Lipofectamine 2000 was used as the transfection reagent. The transfection system for each flask of cells is shown in Table 11.

[0079] Table 11

[0080] Transfection system Volume (μL / well) plasmids 1 μg Lipofectamine 2000 1.5 μL <![CDATA[Opti-MEM TM Medium]]> 100 μL

[0081] H37Rv was cultured in 7H11 medium at 37°C and 5% CO2. After the culture reached the logarithmic phase, infection was performed at an MOI of 10 (cell number: bacterial number = 1:10). Before infection, the cells were counted. In a 12-well plate, the number of RAW264.7 cells was approximately 4×10 5 pcs / hole. Then H37Rv needs 4×10 6 Take 100 μL of H37Rv bacterial solution and measure OD in UV spectrophotometer. 600 The absorbance value is 0.5, and the concentration of Mycobacterium tuberculosis obtained from previous experiments - OD 600 The concentration of the bacterial solution was calculated from the absorbance standard curve to be 8×10 7 If the calculated number of cells / mL is 0.05, 50 μL of H37Rv bacterial solution should be added to each well of RAW264.7 cells. Add the calculated amount of bacterial solution to the wells and incubate at 37°C for 4 hours. Wash three times with PBS to remove uninfected bacteria. Add DMEM containing 50 μg / mL gentamicin and continue culturing the cells for 24 and 36 hours before collecting samples.

[0082] 2) CFU assay after infection with RAW264.7 cells. The specific steps are as follows:

[0083] After the attack, the cells were washed 3-5 times with PBS, and 1 mL of 0.25% Triton X-100 in PBS was added to lyse the cells. Lyse for 10 min at room temperature and dilute the lysate to 10 -2 , 10 -3 , 10 -4After doubling, 100 μL of each sample was taken and plated on 7H11 solid culture medium, and then cultured in a 37°C incubator for 20 days before counting the number of single colonies.

[0084] The results are as follows Figure 3 As shown in the figure, at 24h and 36h after infection, the intracellular bacterial counts of cells transfected with bSP110a and bSP110b were significantly decreased compared with those of cells transfected with bSP110c, indicating that the anti-tuberculosis ability of bSP110c on macrophages was weaker than that of bSP110a and bSP110b.

[0085] S4.2: After 24 hours of expression in the RAW264.7 cell line transfected in step S4.1, the cells were infected with Mycobacterium tuberculosis H37Rv at an MOI of 10 for 36 hours and the cell supernatants were collected and the concentrations of IL6, IL10, IL1β, and TNFα were measured using ELISA kits as follows:

[0086] 1) Sample collection

[0087] The cell transfection and Mycobacterium tuberculosis infection steps were as above. 36 hours after Mycobacterium tuberculosis infection, the cell supernatant was collected, filtered through 0.22 μm, and then tested separately. The supernatant was stored at -80°C during this period.

[0088] 2) Sample testing

[0089] The supernatant of RAW264.7 cells was detected using the Mouse IFN-β ELISA Kit. The detection steps are as follows:

[0090] ① Preparation of standard curve of cell culture supernatant samples

[0091] Take 230 μL of the concentrated standard and add 230 μL of cell culture medium to set the highest concentration for the standard curve (1,000 pg / mL). Add 230 μL of cell culture medium to each tube. Make 1:1 serial dilutions using the high-concentration standard. Ensure thorough mixing between each pipetting. Use cell culture medium as the zero concentration for the standard curve.

[0092] ② Sample addition: Add 100 μL of a 2-fold serial dilution of the standard to the standard wells. Add 100 μL of cell culture medium to the blank wells and 100 μL of the collected cell culture supernatant to the sample wells.

[0093] ③ Add detection antibody: Add 50 μL of detection antibody working solution to each well. Ensure continuous addition without interruption. Complete the addition process within 15 minutes. Seal the plate with sealing film, shake at 100-300 rpm for 2 minutes, and incubate at room temperature (25°C ± 3°C) for 2 hours. After incubation, discard the liquid and wash the plate six times by adding 300 μL of wash solution to each well.

[0094] ④ Enzyme incubation: Add 100 μL of streptavidin working solution to each well and seal the plate with a new sealing film. Oscillate at 100-300 rpm for 2 minutes, incubate at room temperature (25°C ± 3°C) for 45 minutes, and wash six times after incubation.

[0095] ⑤ Add substrate for color development: Add 100 μL of color development substrate to each well, protect from light, and incubate at room temperature (25℃±3℃) for 5-30 minutes.

[0096] ⑥ Add stop solution: Add 100 μL of stop solution to each well. The color will change from blue to yellow.

[0097] ⑦ Detection reading: Within 30 minutes, use a microplate reader to perform dual-wavelength detection and measure the OD value at the maximum absorption wavelength of 450nm.

[0098] The results are as follows Figure 4 As shown, compared with bSP110c, bSP110a and bSP110b promoted the expression of inflammatory factors during H37Rv infection of macrophages, thereby affecting the anti-tuberculosis ability of macrophages.

[0099] Example 5

[0100] The steps for constructing the minigene eukaryotic expression vector of the bSP110 gene are as follows:

[0101] S5.1. Cloning of bovine SP110 exon 1 to exon 11 (excluding the intervening intron, the sequence is shown in SEQ ID NO. 14), intron 11 (the sequence is shown in SEQ ID NO. 15), exon 12 to exon 14 (including the intervening intron, the sequence is shown in SEQ ID NO. 16), intron 14 (the sequence is shown in SEQ ID NO. 17), and exon 15 to exon 19 (excluding the intervening intron) and the 3′ UTR sequence (the sequence is shown in SEQ ID NO. 18)

[0102] 1) Cloning of bovine SP110 exon 1 to exon 11 (excluding the intervening introns)

[0103] The cDNA obtained in S1.2 of Example 1 was used as a PCR template. PCR was performed using the primers shown in Table 12, the system shown in Table 13, and the amplification program shown in Table 14. The reaction products were subjected to agarose gel electrophoresis and recovered using a kit for later use.

[0104] SEQ ID NO.14:

[0105]

[0106] SEQ ID NO.15:

[0107]

[0108] SEQ ID NO.16:

[0109]

[0110] SEQ ID NO.17:

[0111] CGCTCCTCTGTCCTCCACTATCTCCCAGAGTTTGCTCAAATTCGAGTCCATTGAGTCCATAATGCTATCTAACCATCCCCTCAGGGTGCTCATATTTGTGTTACGGACAGTAGCGGGTGGTGGCAGGGTCCCCTCTTGGTTGTAACTGACTCTCAGTTGCTTTGAGTGGTGGTCTACCTGACCACCCACCATCCGTCTTGGGGAGAAAGGACCACAGTGGGGCACTTTGGCACCCCTTCTCCTCCTCCTCCTCCTTTCTCTTCAGTCTTTCCCAGCACCACCTGAATCTTTGTCCAACTGTAAGGATCATGCCTCTCACATAGTTACTTGTAAAACTTAATAGGTTACATTTTACAGCCACAAGAAAGACCCCTATGGATCCTCAGCACACACCCCGGCCCCTAACCAAACAACCCATTGACACAGCAGCTCTGTGTCTTTGTGTATGTCCCTCCGTGTGTCAGTATAACCTCAGGGACTGTTCTCAGAGTTGCCCACGAGCTTACCTGGGCCTGCGCGCAGCGGCTGAGAGCATCTCTGGGCCAGGCCTGGGAAGAGGGAGCAACCTGTCTCAGGTACCAATCCCCTTCTAGCAGGAAAACTCCAAGGAATGTG

[0112] SEQ ID NO.18:

[0113] AGCAGGAAAACTCCAAGGAATGTGCGGTGTGCTGTAAAGGGGGCTCGCTGCTCCGCTGTGACACTTGCCCGCGATTCTTTCACGAGGACTGTCACATCCCACCCGCGGAGGCTGAGAGGAGCCCATGGAGTTGCACCTTCTGCAGGATGAAGGAGTCTTCAGGAAACCAGCAGGGTCTCCAGGAATCTGAGGTTTGGGCAAGGTCCATGCAGCTGGAGGAGCAGCTGAAATGTGAATTTCTCCTCTTGAAAGCCTACTGTCATCCACAAAGCTCTTTCTTTGCGGAGACCCCATGTAATATTCGAGATTATGGTGAGCCCTTTAGGGAAGCCATGTGGTTGGACCTGGTTAAAGAAAGGCTGACTGAGAAAGTGTACACAATGGCTTGGTTTTTACGGGACATGCGCCTAATATTTCACAACCATAAAACATTTTACAAGGCTTCCGACTTTGGTCAGGTAGGACTGGATTTAGAGGCAGAATTTGAAAAGGACCTCAAAGAAGTGCTTATTTGTCATGAAGTCAGTGACAACAGCTTCCAGGCTCATCCTTGATCCTGTTCTGGATGGACTAAAAGCATCCCAACTTCAGGATCCATATAATGGTACCCTGGCCATCCTTAGATTGCCAACGACTCTGGGATGCCATTGGCTGCCCTCGTCATTCAAATCCAACTTTACATATAATCATCATGTCCTCCACCTCCTTTTACTCTTTTTGTATTTCACTCCCCCCTCAAAAAAAAAATTGAATTGTTTCTTAATACCATTAAATACCTCACCACCGTTCACATTCA

[0114] Table 12

[0115]

[0116]

[0117] Table 13

[0118]

[0119] Table 14

[0120]

[0121] 2) Cloning of the 11th intron (5' partial sequence) of bovine SP110

[0122] Using the whole bovine genome as a template, PCR was performed using the primers shown in Table 15, the system shown in Table 13, and the amplification program shown in Table 14. The reaction products were subjected to agarose gel electrophoresis and recovered using a kit for later use.

[0123] Table 15

[0124]

[0125] 3) Cloning of bovine SP110 intron 11 (3' end partial sequence), bovine SP110 exons 12 to 14 (including the middle intron), and bovine SP110 intron 14 (5' end partial sequence)

[0126] Using the whole bovine genome as a template, PCR was performed using the primers shown in Table 16, the system shown in Table 13, and the amplification program shown in Table 14. The reaction products were subjected to agarose gel electrophoresis and recovered using a kit for later use.

[0127] Table 16

[0128]

[0129] 4) Cloning of the 14th intron (3' end partial sequence) of bovine SP110

[0130] Using the whole bovine genome as a template, PCR was performed using the primers shown in Table 17, the system shown in Table 13, and the amplification program shown in Table 14. The reaction products were subjected to agarose gel electrophoresis and recovered using a kit for later use.

[0131] Table 17

[0132]

[0133] 5) Cloning of bovine SP110 exon 15 to exon 19 (excluding the intervening intron) and 3'UTR sequence

[0134] The cDNA obtained in S1.2 of Example 1 was used as a PCR template. PCR was performed using the primers shown in Table 18, the system shown in Table 13, and the amplification program shown in Table 14. The reaction products were subjected to agarose gel electrophoresis and recovered using a kit for later use.

[0135] Table 18

[0136]

[0137] S5.2. Perform agarose gel electrophoresis on the PCR product and purify and recover it using a kit according to the attached instructions. Then, use a homologous recombination kit to mix the system shown in Table 19. Incubate at 50°C for 15 minutes, cool on ice for 1 minute, and use the resulting product directly for transformation.

[0138] Table 19

[0139] Components volume 2×BasicAssemblyMix 5μL PCR products 0.06 pmol pCMV-Flag-N linearized vector 0.03 pmol Nuclease-free Water to10μL

[0140] S5.3. Transform the constructed recombinant plasmid into competent E. coli DH5α strain. After ampicillin screening, single clones were selected and sent to the company for sequencing. The recombinant vector with correct sequencing was named pCMV-Flag-ASVV. The schematic diagram is shown in the figure. Figure 5 As shown, the positive recombinant vector was added with glycerol and stored at -80°C.

[0141] Example 6

[0142] The bovine SP110 gene splicing-edited vector, Replace human / horse SP110 elementMinigene, was constructed by replacing the human or horse SP110 pre-SAND exon and its adjacent 5′-end intron 12 sequence with the 3′-end sequence of bovine SP110 exon 11 based on pCMV-Flag-ASVV. The replaced vector is abbreviated as pCMV-Flag-Rhuman / horse-ASVV. The construction steps are as follows:

[0143] S6.1. Clone the human and horse pre-SAND exon sequence and its adjacent 5′-end 12th intron partial sequence (SEQ ID NO.1 and SEQ ID NO.2). Use the primers shown in Table 21, the human and horse genomes as templates, respectively, to perform PCR using the system shown in Table 13 and the amplification program shown in Table 14. After agarose gel electrophoresis, the reaction products were recovered using a kit to obtain the human and horse pre-SAND exon sequences.

[0144] Table 20

[0145]

[0146] The human pre-SAND exon sequence and its adjacent 5′ end 12th intron sequence are SEQ ID NO.1:

[0147] GAAAAGAAAAAAGGAGAAAGATATCTGTTCAAGCTCAAAAAGGAGATTTCAGAAAAATATTCACCGAAGAGGTAAGCAAGCAAGCCACAGCGCCTCCCCACTGGGCTCCCCAGAGCCCTGGCCTCTGCTGGAAGCTCCACCCGCCCTCTCCTTCTTCTCT CTCCCTTTCCTCCAAAGTTGTTCTCCACCGTCATGGGTACCAGCTGAATTGGGTGCATCAGGAGTGCTGCCAGCCTAACCCACCCCCTTGTGCATGGTGGCCATTTCCATACTTGGAAGGATTCTTTAACACAGAGCTATACAAAGACCCAAAAATGAT

[0148] The horse pre-SAND exon sequence and its adjacent 5′ end 12th intron sequence are SEQ ID NO.2:

[0149] GAAAAGATGAGAGAAAAATGTAGCCAGTCAAGTCCACAAAAAGGGCTGTCAAGAGGTAAGCACAAAGCACGAGGTCCTTACTGCCTCGGTGCTCATCTGGGTCATCACTTGGCCAACCAGCATGCCCTCCTCCCCAGCCACACGGCCACCCG GGTATCACTGGAGCACGGCAGCCCCACCCCTGCCCCCAAGCCACCCAGTCAAAGGACTCCTGTTCACGAAAGAAGGACGACCCAGATGCCATGCTGGTTCAAACAAGCATCTTTTCTCTTATCACTGGGAAGGAGTGGAGGTGGGGGTGGAC

[0150] S6.2. Cloning of the bovine SP110 gene

[0151] Using the pCMV-Flag-ASVV vector constructed in Example 4 as a template, PCR was performed with the primers shown in Table 22, the system shown in Table 13, and the amplification program shown in Table 14. The reaction products were subjected to agarose gel electrophoresis and recovered using a kit to obtain two bovine SP110 gene sequences.

[0152] Table 21

[0153]

[0154] S6.3. Construction of pCMV-Flag-Rhuman / horse-ASVV reporter vector

[0155] The three purified DNA fragments obtained in steps S6.1 and S6.2 were linked into the pCMV-Flag-N backbone vector through the principle of homologous recombination, and then transformed into the competent Escherichia coli DH5α strain. Single colonies were selected by ampicillin screening, and single colonies were picked and propagated in liquid LB medium for sequencing analysis. The obtained recombinant positive plasmids derived from the human SP110 gene were named pCMV-Flag-Rhuman-ASVV, and the recombinant positive plasmids derived from the horse SP110 gene were named pCMV-Flag-Rhorse-ASVV. The vector schematic diagram is shown in FIG. Figure 6 As shown, the positive plasmids with correct sequencing were stored at -80°C for subsequent experiments.

[0156] Example 7

[0157] The bovine SP110 gene splicing-edited vector Replace Knock-in human / horse SP110element Minigene was constructed. That is, the human or horse SP110 pre-SAND exon sequence was replaced with the 3′ end sequence of the bovine SP110 exon 11 based on pCMV-Flag-ASVV, and the adjacent 5′ end 12th intron was inserted thereafter. The replaced vector is abbreviated as: pCMV-Flag-RKIhuman-ASVV or pCMV-Flag-RKIhorse-ASVV. The construction steps are as follows:

[0158] S7.1. Clone the human and horse pre-SAND exon sequences and their adjacent 5′-end 12th intron sequences, following the same steps as S6.1.

[0159] S7.2. Cloning of bovine SP110 gene

[0160] Using the pCMV-Flag-ASVV vector constructed in Example 4 as a template, PCR was performed with the primers shown in Table 23, the system shown in Table 13, and the amplification program shown in Table 14. The reaction products were subjected to agarose gel electrophoresis and recovered using a kit to obtain two bovine SP110 gene sequences.

[0161] Table 22

[0162]

[0163] S7.3. Construction of pCMV-Flag-RKIhuman / horse-ASVV reporter vector

[0164] The three purified DNA fragments obtained in steps S7.1 and S7.2 were linked into the pCMV-Flag-N backbone vector through the principle of homologous recombination, and then transformed into the competent Escherichia coli DH5α strain. Single colonies were selected by ampicillin screening, and single colonies were picked and propagated in liquid LB medium for sequencing analysis. The obtained recombinant positive plasmids derived from the human SP110 gene were named pCMV-Flag-RKIhuman-ASVV, and the recombinant positive plasmids derived from the horse SP110 gene were named pCMV-Flag-RKIhorse-ASVV. The vector schematic diagram is shown in FIG. Figure 7 As shown, the positive plasmids with correct sequencing were stored at -80°C for subsequent experiments.

[0165] Example 8

[0166] To investigate the effect of splicing editing of the bovine SP110 gene on bSP110c production, the steps are as follows:

[0167] S8.1. pCMV-Flag-ASVV and pCMV-Flag-Rhuman / horse-ASVV constructed in Examples 4 and 5 were transfected into NIH3T3 cells, BFF cells, THP-1 cells, and HEK293T cells, respectively. Lip2000 liposome transfection was used (the transfection method was the same as in Example 4 S4.1). The cells were collected 36 hours later, and the total RNA of the collected cells was extracted by the Trizol method. The total RNA was then reverse transcribed into cDNA using a reverse transcription kit according to the attached instructions (the method was the same as in Example 5 S5.4).

[0168] S8.2. Using the cDNA obtained in step S7.1 as a template and the sequences shown in Table 24 as primers, perform fluorescence quantitative PCR (same steps as in Example 5 S5.4) to detect the level of SP110c mRNA.

[0169] Table 23

[0170] Primer name Primer sequence (5′-3′) qPCR-Vc-1-F CCGGGAAAAAGCGAGCAGCCTCATC(SEQ ID NO.45) qPCR-Vc-1-R TCACTTTAGATCTACATCACCTGTG(SEQ ID NO.46) qPCR-Vc-2-human-R TTCTCCTCCAAAGTTGTTCTC(SEQ ID NO.47) qPCR-Vc-2-horse-R CTGGAGCACGGCAGCCCCACCCCT(SEQ ID NO.48)

[0171] The location of SP110 quantitative primer is shown in Figure 8 .

[0172] The results are as follows Figure 9 As shown in the figures, the pCMV-Flag-Rhuman-ASVV and pCMV-Flag-Rhorse-ASVV recombinant reporter vectors constructed in Example 5 can effectively reduce the expression level of the transcript SP110c with weaker anti-tuberculosis ability, while increasing the expression levels of transcripts SP110a and SP110b with stronger anti-tuberculosis ability, and the trends are consistent in different cell lines.

[0173] Embodiment 9

[0174] The effects of the overexpression vectors constructed in Examples 4 and 5 on the anti-tuberculosis ability were investigated in the following steps:

[0175] First, the RAW264.7 cell line was transfected with pCMV-Flag-ASVV, pCMV-Flag-Rhuman-ASVV, and pCMV-Flag-Rhorse-ASVV using Lip2000 (each plasmid was transfected into two 60 cm culture dishes of cells). H37Ra tuberculosis was then infected at an MOI of 10. Cells were lysed 12 and 72 hours after infection, and the cell lysates were collected and plated onto 7H11 solid medium. Culture was continued for 3 weeks before CFU analysis. (The same procedures as in Example 4, S4.1)

[0176] The results are as follows Figure 10 As shown, at 12 hours of infection, the number of viable intracellular bacteria in cells transfected with pCMV-Flag-ASVV and pCMV-Flag-Rhuman / Rhorse-ASVV was not significantly different. At 72 hours of infection, the number of intracellular bacteria in cells transfected with pCMV-Flag-Rhuman-ASVV and pCMV-Flag-Rhorse-ASVV was significantly lower than that at 12 hours of infection. The number of intracellular bacteria in cells transfected with pCMV-Flag-ASVV was also significantly lower than that at 12 hours of infection, indicating that the overexpression reporter vectors can inhibit the growth of Mycobacterium tuberculosis to a certain extent. At 72 hours of infection, the number of intracellular bacteria in cells transfected with pCMV-Flag-Rhuman-ASVV and pCMV-Flag-Rhorse-ASVV was significantly lower than that in cells transfected with pCMV-Flag-ASVV.

[0177] Splicing editing of the bovine SP110 gene can significantly reduce the level of SP110c transcripts and significantly increase the abundance of SP110a and SP110b transcripts, thereby enhancing the ability of macrophages to fight Mycobacterium tuberculosis infection.

[0178] Therefore, the present invention can effectively improve the anti-tuberculosis ability of cattle by introducing the 12th exon of the human or horse SP110 gene and the adjacent 5′-end 12th intron partial sequence (pre-SAND-exon sequence) into the bovine SP110 gene; the provided bovine SP110 gene splicing editing site can be used as a bovine tuberculosis resistance linkage site for breeding tuberculosis-resistant cattle, which can effectively simplify the breeding method, improve the breeding efficiency, and save the breeding cost.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of bovine SP110 gene splicing and editing body, vector or engineered bacteria containing bovine SP110 gene splicing and editing body in the preparation of anti-bovine tuberculosis preparation, characterized in that: The splicing-edited body is a recombinant nucleic acid obtained by replacing the 3'-terminal partial sequence of exon 11 of bovine SP110 with the pre-SAND exon of human or equine SP110 gene and the partial sequence of its adjacent 5'-terminal intron 12, wherein the pre-SAND exon of human SP110 gene and the partial sequence of its adjacent 5'-terminal intron 12 are as shown in SEQ ID NO.1, and the pre-SAND exon of equine SP110 gene and the partial sequence of its adjacent 5'-terminal intron 12 are as shown in SEQ ID NO.2; the primers for amplifying bovine SP110 gene are bSP110-exon1-F and bSP110-3UTR-R; the sequence of bSP110-exon1-F is as shown in SEQ ID NO.19, and the sequence of bSP110-3UTR-R is as shown in SEQ ID NO.28; The 3'-terminal partial sequence of exon 11 of bovine SP110 is the fragment between primers horse-bSP110-exon11-overlap-R and horse-bSP110-intron11-overlap-F5 or hunman-bSP110-exon11-overlap-R and hunman-bSP110-intron11-overlap-F5 in bovine SP110; The sequence of horse-bSP110-exon11-overlap-R is as shown in SEQ ID NO.35; the sequence of horse-bSP110-intron11-overlap-F5 is as shown in SEQ ID NO.37; the sequence of hunman-bSP110-exon11-overlap-R is as shown in SEQ ID NO.34; the sequence of hunman-bSP110-intron11-overlap-F5 is as shown in SEQ ID NO.

36.

2. Use of the bovine SP110 gene splicing-edited body, vector or engineered bacterium containing the bovine SP110 gene splicing-edited body as described in claim 1 in the breeding of tuberculosis-resistant dairy cows.

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