Application of splicing and editing of cattle SP110 gene in enhancing anti-tuberculosis ability of cattle

Abstract

The invention discloses an application of cattle SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle, and belongs to the technical field of cattle anti-tuberculosis breeding, starting from splicing related cis-acting elements, the variable splicing reaction of the cattle SP110 is regulated by introducing the cis-acting elements, so that the anti-tuberculosis ability of the cattle is influenced. The sequence of the cis-acting element is named as a pre-SAND-exon sequence, the cis-acting element is located on the 12th exon of a human or horse SP110 gene and the 12th intron adjacent to the 5'end of the human or horse SP110 gene, and the sequences of the cis-acting element are shown as SEQ ID NO.1 and SEQ ID NO.2. According to the invention, the pre-SAND-exon sequence is introduced into the cattle SP110 gene for the first time, so that the variable splicing of the cattle SP110 gene is regulated and controlled, the expression abundance of a splicing body bSP110c with relatively low anti-tuberculosis capability is reduced, and the abundance of bSP110a and bSP110b splicing bodies with anti-tuberculosis infection capability is regulated and controlled to be higher. The provided cattle SP110 gene splicing editing can effectively improve the anti-tuberculosis ability of cattle, can be used for breeding 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 anti-tuberculosis breeding, and in particular to the application of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of bovine. Background Art

[0002] Bovine tuberculosis (BTB) is a chronic, consumptive zoonosis caused primarily by infection with Mycobacterium bovis (M. bovis). According to statistics, there are about 50 million tuberculosis-positive cattle worldwide each year, causing agricultural losses of about $3 billion each year. In addition, about 10%-15% of human tuberculosis infections are caused by bovine tuberculosis, threatening animal-derived food safety and public health safety. In recent years, the scale of my country's cattle industry has continued to expand. While meeting the supply of animal products, dairy cow tuberculosis has become a key issue affecting the sustainable development of my country's cattle 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-mentioned purpose, the present invention provides the application of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle, wherein the splicing editing is achieved by introducing a cis-acting element, and 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 alternative 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 introducing a cis-acting element upstream of the SP110 gene to regulate the alternative splicing of bovine SP110, 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 editing as described above, and the vector or engineered bacteria containing the bovine SP110 gene splicing 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 can significantly reduce the abundance of bovine bSP110c transcripts and increase the abundance of bSP110a and bSP110b transcripts by introducing a cis-acting element (pre-SAND-exon sequence) into the bovine SP110 gene, thereby increasing the expression of inflammatory factors during the infection of macrophages by Mycobacterium tuberculosis, reducing the intracellular proliferation ability of Mycobacterium tuberculosis, and improving the 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 introduces 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 to regulate the splicing and editing of the bovine SP110 gene, thereby effectively improving the anti-tuberculosis ability of the bovine;

[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 accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

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

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

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

[0020] Figure 4 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 elementMinigene 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] Fig. 9 The mRNA levels of the three transcripts of the bovine SP110 gene expressed by the three minigene reporter vectors constructed in Example 5, Example 6 and Example 7 in Example 8 of the present invention;

[0026] Fig.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 through the accompanying drawings and embodiments.

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meanings as those generally understood by those skilled in the art to which the present application belongs, and experimental methods not specifically described are conventional experimental methods in the art.

[0029] The bovine SP110 gene splicing editing provided by the present invention can reduce the generation amount of a transcript bSP110c (amino acid sequence as shown in SEQ ID NO.9) of the bovine SP110 gene without anti-tuberculosis function by regulating the alternative 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 the 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 all obtained through commercial channels.

[0037] Embodiment 1

[0038] The three transcripts of 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. The total RNA of bovine macrophages was extracted by Trizol method. The first reverse transcription was performed. The reverse transcription system was shown in Table 2. The reaction conditions were: 72℃ for 3 min, 42℃ for 2 min. Then it was quickly placed on ice for the second reverse transcription. The system was shown in Table 3. The reaction conditions were: 42℃ for 90 min, 70℃ for 10 min. cDNA was obtained. A part of it was used for the next experiment, and the rest was frozen at -80℃ for later use.

[0043] Table 2

[0044] Components Volume RNASample 1 μg 3'-CDSprimerA 1μL RandomPrimer 1μL <![CDATA[RNaseFreeddH2O]]> To12μL Total volume 12μL

[0045] Table 3

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

[0047] S1.3, using the system shown in Table 4 and the amplification conditions shown in Table 5, the cDNA obtained in step S1.2 was used as a template to perform nested PCR cloning of SP110 transcripts. Using the system shown in Table 6 and the amplification conditions shown in Table 7, the next step of nested PCR was performed using the first step reaction product as a template. The PCR products were separated by agarose gel electrophoresis, and the amplification results were as follows: Figure 1 As shown, all bands were cut out and recovered and purified using an agarose gel recovery kit.

[0048] Table 4

[0049] Component Volume <![CDATA[PCR-GradeH2O]]> 15.5μL 2×SeqAmpBuffer 25μL SeqAmpDNAPolymerase 1μL template 2.5μL 10×UPM 5μL SP110-3'RACE-NGSP 1μL 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, the recovered product obtained in step S1.3 was prepared into a ligation system by using the components shown in Table 8, mixed well, and ligated at 4°C overnight. The ligation product was then transformed into Escherichia coli DH5α, and then single colonies were screened by ampicillin, and single colonies were picked and propagated in liquid LB medium by shaking. When the OD was 0.6, the plasmid was extracted using a plasmid extraction kit.

[0058] Table 8

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

[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, and their amino acid sequences were SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0061] Embodiment 2

[0062] The three transcripts obtained in Example 1 were subjected to domain analysis in the following manner:

[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. A schematic diagram of the functional domains of the bSP110 gene was drawn based on the analysis results. 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] Embodiment 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 in 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 main SP110 spliceosomes bSP110a, bSP110b, and bSP110c, respectively. The primer sequences are shown in Table 9 (lowercase represents the introduced restriction site, F represents the upstream primer, and R represents the downstream primer sequence):

[0067] Table 9

[0068]

[0069]

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

[0071] Table 10

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

[0073] Embodiment 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, and infected with H37Rv Mycobacterium tuberculosis after 24 hours of expression, as follows:

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

[0077] Mycobacterium tuberculosis strains H37Rv and M. bovis were from the research group of Guo Aizhen at Huazhong Agricultural University. The whole process of this bacterial attack experiment was carried out in the animal biosafety level 3 laboratory of Huazhong Agricultural University.

[0078] First, the constructed eukaryotic expression vectors pCMV-HA-bSP110a, pCMV-HA-bSP110b and pCMV-HA-bSP110c were used to transfect RAW264.7 cells in 12-well plates, and the cells were cultured to a confluence of 70% before transfection. The liposome transfection method was used, and the transfection reagent was Lipofectamine 2000. The transfection system of each bottle of cells is shown in Table 11.

[0079] Table 11

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

[0081] H37Rv was cultured in 7H11 medium at 37°C and 5% CO2. After the logarithmic period, the cells were infected 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 / mL, then 50μL H37Rv bacterial solution should be added to each well of RAW264.7 cells. Add the calculated bacterial solution into the cell wells, incubate at 37℃ for 4h, then wash with PBS 3 times to remove the uninfected bacteria, add DMEM cell culture medium containing 50μg / mL gentamicin and continue to culture the cells for 24h and 36h before collecting samples.

[0082] 2) CFU experiment after RAW264.7 cells were challenged with bacteria. 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% TritonX-100 in PBS was added to lyse the cells. Lysis was carried out at room temperature for 10 min, and the lysate was diluted to 10 -2 , 10 -3 , 10 -4After doubling, take 100 μL of each plate and spread it on 7H11 solid culture medium, place it in a 37°C incubator and culture it for 20 days, then count the number of single colonies.

[0084] The results are as follows Figure 3 As shown, at 24h and 36h of infection, the number of intracellular bacteria transfected with bSP110a and bSP110b was significantly decreased compared with that of bSP110c, indicating that the anti-tuberculosis ability of bSP110c on macrophages is weaker than that of bSP110a and bSP110b.

[0085] S4.2: After the RAW264.7 cell line was transfected and expressed for 24 hours in step S4.1, the cell line was infected with Mycobacterium tuberculosis H37Rv at 10 moi for 36 hours, and the cell supernatant was collected and the concentrations of IL6, IL10, IL1β, and TNFα were detected using an ELISA kit. The steps are as follows:

[0086] 1) Sample collection

[0087] The steps of cell transfection and infection with Mycobacterium tuberculosis were as above. After 36 hours of infection with Mycobacterium tuberculosis, the cell supernatant was collected and tested after filtering through 0.22 μm. 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 as the highest concentration for the standard curve (1,000 pg / mL). Add 230 μL of cell culture medium to each tube. Make a 1:1 serial dilution with the high concentration standard. Make sure to mix thoroughly each time you pipette. Use cell culture medium as the zero concentration for the standard curve.

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

[0093] ③ Add detection antibody: Add 50μL detection antibody working solution to each well. Ensure continuous sample addition without interruption. The sample addition process should be completed within 15 minutes. Then seal the plate with a sealing film, shake at 100-300r / min for 2 minutes, and incubate at room temperature (25℃±3℃) for 2 hours. After incubation, discard the liquid, add 300μL washing solution to each well to wash the plate, and wash 6 times.

[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 r / min for 2 minutes, incubate at room temperature (25℃±3℃) for 45 minutes, and wash 6 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] ⑥Add stop solution: Add 100 μL of stop solution to each well. The color changes from blue to yellow.

[0097] ⑦ Detection reading: Within 30 minutes, use an ELISA 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] Embodiment 5

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

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

[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, and 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 then 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 (partial sequence at the 5' end) of bovine SP110

[0122] Using the whole genome of cattle 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 then recovered using a kit for later use.

[0123] Table 15

[0124]

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

[0126] Using the whole genome of cattle 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 then 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 genome of cattle 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 then recovered using a kit for later use.

[0131] Table 17

[0132]

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

[0134] The cDNA obtained in S1.2 of Example 1 was used as a PCR template, and 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 then recovered using a kit for later use.

[0135] Table 18

[0136]

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

[0138] Table 19

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

[0140] S5.3. Transform the constructed recombinant plasmid into the competent DH5α strain of E. coli. After ampicillin screening, single clones were selected and sent to the company for sequencing. The positive 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] Embodiment 6

[0142] The vector Replace human / horse SP110 elementMinigene was constructed after splicing and editing of bovine SP110 gene, that is, the human or horse SP110 pre-SAND exon and its adjacent 5′ end 12th intron partial sequence were replaced with the 3′ end partial sequence of bovine SP110 exon 11 on the basis of pCMV-Flag-ASVV. The replaced vector is referred to as pCMV-Flag-Rhuman / horse-ASVV, and 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, and use 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 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 partial sequence are SEQ ID NO.1:

[0147] GAAAAGAAAAAAGGAGAAAGATATCTGTTCAAGCTCAAAAAGGAGATTTCAGAAAAATATTCACCGAAGAGGTAAGCAAGCAAGCCACAGCGCCTCCCCACTGGGCTCCCCAGAGCCCTGGCCTCTGCTGGAAGCTCCACCCGCCCTCTCCTTCTTCTCT CTCCCTTTCCTCCAAAGTTGTTCTCCACCGTCATGGGTACCAGCTGAATTGGGTGCATCAGGAGTGCTGCCAGCCTAACCCACCCCCTTGTGCATGGTGGCCATTTCCATACTTGGAAGGATTCTTTAACACAGAGCTATACAAAGACCCAAAAATGAT

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

[0149] GAAAAGATGAGAGAAAAATGTAGCCAGTCAAGTCCACAAAAAGGGCTGTCAAGAGGTAAGCACAAAGCACGAGGTCCTTACTGCCTCGGTGCTCATCTGGGTCATCACTTGGCCAACCAGCATGCCCTCCTCCCCAGCCACACGGCCACCCG GGTATCACTGGAGCACGGCAGCCCCACCCCTGCCCCCAAGCCACCCAGTCAAAGGACTCCTGTTCACGAAAGAAGGACGACCCAGATGCCATGCTGGTTCAAACAAGCATCTTTTCTCTTATCACTGGGAAGGAGTGGAGGTGGGGGTGGAC

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

[0151] Using the pCMV-Flag-ASVV vector constructed in Example 4 as a template, PCR was performed using 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 segments of 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 connected to 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, and single colonies were picked to liquid LB medium for shaking and propagation, and then sent for sequencing analysis. The recombinant positive plasmid derived from the human SP110 gene was named pCMV-Flag-Rhuman-ASVV, and the recombinant positive plasmid derived from the horse SP110 gene was named pCMV-Flag-Rhorse-ASVV. The vector schematic diagram is shown in the figure. Figure 6 As shown, the positive plasmid with correct sequencing was stored at -80°C for subsequent experiments.

[0156] Embodiment 7

[0157] The vector Replace Knock-in human / horse SP110element Minigene was constructed after splicing and editing of bovine SP110 gene. That is, the partial sequence of human or horse SP110 pre-SAND exon was replaced with the partial sequence of the 3′ end of the 11th exon of bovine SP110 on the basis of pCMV-Flag-ASVV, and the adjacent 5′ end 12th intron was inserted thereafter. The replaced vector was 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 the adjacent 5′-end 12th intron partial sequence, 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 using 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 segments of 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 connected to 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, and single colonies were picked to liquid LB medium for shaking and propagation, and then sent for sequencing analysis. The obtained recombinant positive plasmid derived from the human SP110 gene was named pCMV-Flag-RKIhuman-ASVV, and the recombinant positive plasmid derived from the horse SP110 gene was named pCMV-Flag-RKIhorse-ASVV. The vector schematic diagram is shown in the figure. Figure 7 As shown, the positive plasmid with correct sequencing was stored at -80°C for subsequent experiments.

[0165] Embodiment 8

[0166] The effect of splicing and editing of bovine SP110 gene on bSP110c production was investigated in the following steps:

[0167] S8.1. The 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. The cells were collected 36 hours after lip2000 liposome transfection (the transfection method was the same as in Example 4 S4.1), and the total RNA of the collected cells was extracted by the Trizol method. Then, the total RNA obtained was 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, fluorescence quantitative PCR was performed (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 Fig. 9 As shown, 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 the transcripts SP110a and SP110b with stronger anti-tuberculosis ability, and the trends in different cell lines are consistent.

[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, pCMV-Flag-ASVV, pCMV-Flag-Rhuman-ASVV and pCMV-Flag-Rhorse-ASVV were transfected into RAW264.7 cell line (two 60 cm culture dishes were transfected with each plasmid) with lip2000, and H37Ra Mycobacterium tuberculosis was infected at MOI=10. The cells were lysed at 12h and 72h of infection, and the cell lysates were collected and plated into 7H11 solid culture medium. After further culture for 3 weeks, CFU analysis was performed. (The steps are the same as those in Example 4 S4.1)

[0176] The results are as follows Fig.10 As shown in the figure, at 12h of infection, the number of intracellular live bacteria in cells transfected with pCMV-Flag-ASVV and pCMV-Flag-Rhuman / Rhorse-ASVV was not significantly different. At 72h of infection, the number of intracellular live bacteria in cells transfected with pCMV-Flag-Rhuman-ASVV and pCMV-Flag-Rhorse-ASVV was significantly reduced compared with the number of live bacteria in cells transfected with pCMV-Flag-ASVV, and the number of intracellular live bacteria in cells transfected with pCMV-Flag-ASVV was also significantly reduced compared with the number of live bacteria in cells transfected with pCMV-Flag-ASVV, indicating that the overexpression reporter vector can inhibit the growth of Mycobacterium tuberculosis to a certain extent. At 72h of infection, the number of intracellular live 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 cattle SP110 gene; the provided bovine SP110 gene splicing editing site can be used as a bovine tuberculosis resistance linkage site for breeding of 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 solution of the present invention rather than to limit it. 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 solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle, characterized by: The splicing editing is achieved by introducing a cis-acting element, wherein 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 from human is shown in SEQ ID NO.1, and the sequence from horse is shown in SEQ ID NO.

2.

2. The use of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle according to claim 1, characterized in that: The application is to regulate the variable splicing of bovine SP110 by introducing cis-acting elements, thereby improving the anti-tuberculosis ability of cattle.

3. The use of bovine SP110 gene splicing editing in enhancing the anti-tuberculosis ability of cattle according to claim 2, characterized in that: 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 the 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.

4. The use of bovine SP110 gene splicing editing in promoting the expression of inflammatory factors as claimed in claim 1, characterized in that: The application is achieved by introducing a cis-acting element upstream of the SP110 gene to regulate the alternative splicing of bovine SP110, thereby reducing the abundance of the transcript bSP110c and increasing the abundance of bSP110a and bSP110b.

5. Use of the bovine SP110 gene splicing editing as claimed in claim 1, or a vector or engineered bacteria comprising the bovine SP110 gene splicing editing in reducing the intracellular proliferation ability of Mycobacterium tuberculosis.

6. Use of the bovine SP110 gene splicing editing as claimed in claim 1, or a vector or engineered bacteria containing the bovine SP110 gene splicing editing in the preparation of an anti-tuberculosis preparation.

7. Use of the bovine SP110 gene splicing editing as claimed in claim 1, and a vector or engineered bacteria comprising the bovine SP110 gene splicing editing in breeding of tuberculosis-resistant dairy cows.

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