Application of lysine in preparation of medicine for retarding increase of BLV (Bromovirus) previrus load in BLV positive bovine lymphocytes

Abstract

The invention relates to application of lysine in preparation of a medicine for alleviating increase of BLV (bovine leukemia virus) previrus load in BLV positive bovine lymphocytes, and belongs to the technical field of bovine leukemia treatment. In order to prevent a cell receptor CAT1 of the BLV from being combined with the BLV or reduce the binding rate of the CAT1 and the BLV so as to block BLV infection, the CAT1 is a Na < + >-independent transporter widely existing on the cell surface and is related to transportation of cationic amino acids (lysine, histidine and arginine), and it is speculated that when the extracellular cationic amino acid concentration is increased, the CAT1 is a Na < + >-independent transporter, the CAT1 is a Na < + >-independent transporter, and the CAT1 is a Na < + >-independent transporter. The BLV and the cationic amino acid have the possibility of competitively binding to CAT1. The influence of the cationic amino acid (Lys) on the BLV pre-virus load in the cow lymphocytes is researched through in-vivo and in-vitro tests, and the result shows that the trend of increasing of the BLV positive bovine pre-virus load can be slowed down by feeding the rumen bypass lysine with the dosage of 50g per day for 30 days.

Description

Technical Field

[0001] The present invention belongs to the technical field of bovine leukemia treatment, and specifically relates to the application of lysine in the preparation of a drug for slowing down the increase in the proviral load of BLV in BLV-positive bovine lymphocytes. Background Art

[0002] Bovine leukemia is a delayed, neoplastic disease caused by the Bovine leukemia virus (BLV). Epidemiologically, this disease can be divided into two major types: Sporadic Bovine Leukosis (SBL) and Enzootic Bovine Leukemia (EBL). The two types of bovine leukemia are mainly characterized by T-cell and B-cell leukemia, respectively (Gillet N, Florins A, Boxus M, et al. Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human [J]. Retrovirology, 2007, 4:18.). BLV mainly causes EBL, and in cattle, the incidence of EBL is much higher than that of SBL (Onuma M, Honma T, Mikami T, et al. Studies on the sporadic and enzootic forms of bovine leukosis [J]. J Comp Pathol, 1979, 89(2):159-167.). BLV mainly leads to a decline in the immune function of infected cattle, subsequently triggering other diseases such as mastitis and gastroenteritis (Baréz PY, de Brogniez A, Carpentier A, et al. Recent Advances in BLV Research [J]. Viruses, 2015, 7(11):6080-6088.) (Frie M C, Coussens P M. Bovine leukemia virus: a major silent threat to proper immune responses in cattle [J]. Vet Immunol Immunopathol, 2015, 163(3-4):103-114.), resulting in a decrease in milk production and an increase in the culling rate of dairy cows, causing huge economic losses to the dairy industry. Most BLV-positive cattle are asymptomatic, but approximately one-third of them develop persistent lymphocytosis (PL) and gradually progress to B-cell leukemia / lymphoma.It has been found that the probability of culling BLV-positive cows is 30% higher than that of negative cows (Benitez O J, LaDronka R M, Norby B, et al. The effect of bovine leukemia virus on dairy cow longevity[J]. JDS Communications, 2022, https: / / doi.org / 10.3168 / jdsc.2021-0187.), which has caused huge losses to the livestock and dairy industries.

[0003] BLV has been widely prevalent worldwide along with the trading of dairy cows, beef cattle and related livestock products. Currently, BLV is well controlled in most Western European countries, but the infection and transmission of BLV are still very common in the Eastern European region ( D, I, M, et al. Identification of a new genotype of bovine leukemiavirus[J]. Arch Virol, 2012, 157(7):1281-1290.)(Rola- M, Pluta A, Olech M, et al. The molecular characterization of bovine leukaemia virus isolates from Eastern Europe and Siberia and its impact on phylogeny[J]. PLoS One, 2013, 8(3): e58705.). In China, due to the characteristics of high infection rate, low incidence rate and low mortality rate of BLV, BLV has not attracted the attention of relevant departments and livestock owners, resulting in a further increase in the infection rate of BLV, which has caused huge economic losses to the animal husbandry and dairy industries in China (Nishimori A, Andoh K, Matsuura Y, et al. Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis[J]. Arch Virol, 2021, 166(3): 841-851.). Therefore, it is urgent to establish a supporting BLV public health risk control strategy to prevent and defuse this potential risk. However, the BLV purification and eradication strategies proposed abroad are not economically applicable to China before, and the specific drugs and vaccines for BLV have not been fully developed yet. Therefore, relying on the existing technology, the virus cannot be completely cleared from the country.

[0004] Cationic amino acid transporters 1 (CAT1) is a member of the solute carrier 7 family (SLC7), with 622 amino acid residues, extremely strong hydrophobicity, and is expressed on the surface of most cells in the body. The expression level varies depending on the cell and tissue, and is related to the Na of lysine (Lys), histidine (His) and arginine (Arg) +It is related to non-dependent transport. In 2019, Professor Bailanlan and his team demonstrated that CAT1 is also a cellular receptor for BLV by constructing the bovine CAT1 (bCAT1) / SLC7A1 expression plasmid and the co-localization experiment of the envelope glycoprotein (Env) of BLV and CAT1 (Bai L, Sato H, Kubo Y, et al. CAT1 / SLC7A1 acts as a cellular receptor for bovine leukemia virus infection [J]. FASEB J, 2019, 33(12): 14516-14527.). Therefore, if the binding of CAT1 to BLV can be blocked or the binding rate of CAT1 to BLV can be decreased, the infection of BLV can be prevented from the adsorption process, thereby reducing the proviral load of BLV.

[0005] Lysine (Lys) is the most restrictive amino acid in animal organisms. It has functions such as enhancing immunity, increasing the appetite of livestock and poultry, participating in fat metabolism, and promoting the growth and development of livestock and poultry. It is essential for various life activities of animals, but it cannot be synthesized in the body and must be obtained from external foods. However, after the ingested amino acids enter the rumen of ruminants, they are easily degraded by the microorganisms in the rumen. Through research, it has been found that the most easily degraded amino acids are Lys and methionine (Bach A, Stern M D. Effects of different levels of methionine and ruminally undegradable protein on the amino acid profile of effluent from continuous culture fermenters[J]. J Anim Sci, 1999, 77(12):3377 - 3384.). To achieve a nutritional balance model and regulate the amino acid balance in the small intestine, scholars have studied the effects of rumen-protected agents on the absorption of amino acids and others. Rumen protection is a protective measure that protects some nutrients through rumen technology to prevent them from being degraded in the rumen and instead being digested and absorbed in the small intestine. Luan Yujing et al. found that by feeding rumen-protected lysine (RPLys), the daily weight gain, digestible nitrogen, deposited nitrogen, etc. of beef cattle can be increased (Luan Yujing, Yang Zaibin, Jiang Shuzhen, et al. Effects of different levels of lysine on the metabolic laws of rumen nutrients in beef cattle[J]. China Herbivores, 2004(04):3 - 5.). Liu Jianxin et al. added RPLys to the diet of Holstein cows for research and found that the milk production of the experimental group of cows increased significantly, and the content of milk protein also increased (Yun Fuyu, Gao Min, Li Manquan, et al. Effects of rumen-protected lysine on milk production and milk composition of dairy cows[J]. Animal Husbandry and Feed Science, 2011, 32(Z1):72 - 74.). Moreover, adding RPLys to the diet of dairy cows can also balance the utilization system of amino acids in the dairy cow body, improve the utilization rate of protein in feed, reduce nitrogen excretion, and protect the environment. Currently, the research on rumen-protected amino acids mainly focuses on RPLys and rumen-protected methionine, and it does not yet involve the use of rumen-protected amino acids for the treatment of bovine leukemia. Summary of the Invention

[0006] To prevent the binding of the cell receptor CAT1 of BLV to BLV or reduce the binding rate of CAT1 to BLV, thereby blocking BLV infection. Based on the fact that CAT1 is a type of Na that widely exists on the cell surface +The non-dependent transporter is related to the transport of cationic amino acids (lysine, histidine, arginine). It is speculated that when the extracellular concentration of cationic amino acids is increased, there is a possibility that BLV competes with cationic amino acids for binding to CAT1. Taking this as a starting point, in vivo and in vitro experiments were conducted to explore the effect of cationic amino acids (Lys) on the proviral load of BLV in dairy cow lymphocytes, providing a scientific theoretical basis for the prevention and treatment of BLV.

[0007] To solve the above technical problems and achieve the corresponding technical effects, the present invention provides the following technical solutions:

[0008] The first object of the present invention is to provide the use of lysine in the preparation of a drug for slowing down the increase in the proviral load of BLV in BLV-positive bovine lymphocytes.

[0009] In one embodiment of the present invention, the lysine is rumen-protected lysine.

[0010] In one embodiment of the present invention, the administration mode of the drug is oral administration.

[0011] In one embodiment of the present invention, the dose of rumen-protected lysine in the drug is 50 g / d.

[0012] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0013] The second object of the present invention is to provide the use of lysine in the preparation of a drug for alleviating bovine leukemia.

[0014] In one embodiment of the present invention, the lysine is rumen-protected lysine.

[0015] In one embodiment of the present invention, the administration mode of the drug is oral administration.

[0016] In one embodiment of the present invention, the dose of rumen-protected lysine in the drug is 50 g / d.

[0017] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0018] The beneficial effects of the present invention:

[0019] The present invention explores the effect of cationic amino acids (Lys) on the BLV proviral load in bovine lymphocytes through in vivo and in vitro experiments. For in vitro experiments, we first tested the effects of different concentrations of Lys on the proliferation of lymphocytes cultured in vitro, and determined that within a certain concentration range, Lys not only has no toxic effect on bovine lymphocytes, but can promote their proliferation, and the promotion effect is strongest at 48 hours. Since the survival time of bovine lymphocytes in vitro is not long, we finally selected Lys concentrations of 0.27 (Ctrl), 3.2, and 12.8 mmol / L and a stimulation time of 48 hours for subsequent cell experiments; secondly, we tested the effects of different concentrations of Lys on the proliferation of bovine lymphocytes cultured in vitro. The expression levels of CAT1 protein and mRNA in lymphocytes and the effect on the BLV proviral load in in vitro cultured bovine lymphocytes were studied. The results showed that the expression levels of CAT1 and mRNA can be regulated by the concentration of Lys, and as the Lys concentration increases, the expression of CAT1 and mRNA also increases; compared with the Ctrl group, the BLV proviral load in the low Lys concentration group and the high Lys concentration group increased significantly, but compared with the low Lys concentration group, the BLV proviral load in the high Lys concentration group decreased significantly. From this, we speculate that high doses of Lys and BLV have a competitive binding effect on CAT1, thus leading to a decrease in viral load. 3.2mmol / L lysine can increase the susceptibility of lymphocytes to BLV by increasing the expression of CAT1 protein, and 12.8mmol / L lysine can reduce the susceptibility of lymphocytes to BLV by competitively binding to CAT1. In vivo experiments were subsequently conducted based on this result to further determine whether Lys really has a competitive inhibitory effect on BLV entry into cells. In the in vivo experiment, the indicators of dairy cows before and after feeding RPLys were tested. The results showed that the contents of lysine, histidine and arginine in lymphocytes of BLV-positive cows were higher than those of BLV-negative cows, but the difference was not significant. Before feeding RPLys, the expression of CAT1 protein in cells of BLV-positive cows showed an upward trend, but the difference was not significant. After feeding RPLys, the expression levels of CAT1 protein and mRNA in lymphocytes of cows in the HPVL group and LPVL group were significantly increased (P<0.05). The digital PCR results showed that the BLV proviral load in the HPVL group and LPVL group first showed an obvious upward trend (0-20 days) and then decreased significantly (20-30 days) during the 0-30 days of feeding RPLys, while the proviral load in the Ctrl group showed a fluctuating upward trend. Judging from the above results, feeding rumen lysine at a dose of 50g per day for 30 days can slow down the trend of the increase in proviral load of BLV-positive cows. This study provides a research basis for a deeper understanding of the role of CAT1 in the BLV infection process and a scientific theoretical basis for the prevention and treatment of BLV. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the standard curve graph of fluorescence quantitative PCR for the full-length BLV plasmid;

[0021] Figure 2 It is the identification result graph of lymphocyte quantity, survival rate and morphology; among them, Figure 2 A in it is the statistical result graph of lymphocyte quantity and survival rate, Figure 2 B in it is the morphology graph of lymphocytes after nuclear staining with DAPI nucleic acid dye under a fluorescence microscope;

[0022] Figure 3 It is the detection result graph of the relative expression levels of CAT1 protein and CAT1 mRNA in lymphocytes under different Lys concentrations; among them, Figure 3 A in it is the western blot band result graph of CAT1 in lymphocytes under different Lys concentrations, Figure 3 B in it is the gray-scale analysis result graph of the expression of CAT1 protein in lymphocytes under different Lys concentrations, Figure 3 C in it is the detection result graph of the relative expression levels of CAT1 mRNA in lymphocytes under different Lys concentrations; * and *** are both markers made by comparing with the data of the Ctrl group, * indicates a significant difference between the two groups (p<0.05), and *** indicates an extremely significant difference between the two groups (p<0.001);

[0023] Figure 4 It is the nucleic acid electrophoresis graph of BLV-infected lymphocytes (pol gene); among them, M is DL-500 Marker, 1 is the negative control (water), 2 is the positive control (pBLV344H), 3 is the DNA sample, and 4 is the cDNA sample;

[0024] Figure 5 It is the result graph of digital PCR detecting the effect of Lys on the BLV proviral load; *** indicates an extremely significant difference (p<0.001);

[0025] Figure 6 It is the analysis result graph of the amino acid content in lymphocytes of BLV-negative, HPVL and LPVL dairy cows; among them, Figure 6 A in it is the analysis result graph of the Lys content in lymphocytes of BLV-negative, HPVL and LPVL dairy cows, Figure 6 B in it is the analysis result graph of the His content in lymphocytes of BLV-negative, HPVL and LPVL dairy cows, Figure 6 C in it is the analysis result graph of the Arg content in lymphocytes of BLV-negative, HPVL and LPVL dairy cows;

[0026] Figure 7Analysis result graph of the relative expression level of CAT1 protein in lymphocytes of BLV-negative, HPVL and LPVL dairy cows; among them, Figure 7 A in Figure 7 is the Western blot band result graph of CAT1,

[0027] Figure 8 Result graph of the effect of RPLys on the expression of CAT1 protein in lymphocytes of HPVL group dairy cows detected by Western blotting; among them, Figure 8 A in Figure 8 is the detection result graph of the Western blot band of CAT1,

[0028] Figure 9 Result graph of the effect of RPLys on the expression of CAT1 protein in lymphocytes of LPVL group dairy cows detected by Western blotting; among them, Figure 9 A in Figure 9 is the detection result graph of the Western blot band of CAT1,

[0029] Figure 10 Result graph of the effect of RPLys on the expression of CAT1 protein in lymphocytes of Ctrl group dairy cows detected by Western blotting; among them, Figure 10 A in Figure 10 is the detection result graph of the Western blot band of CAT1,

[0030] Figure 11 Result graph of the effect of RPLys on the relative expression level of CAT1 mRNA in lymphocytes of different experimental groups of dairy cows detected by RT-qPCR; *** indicates extremely significant difference (p < 0.001), ns indicates no significant difference (p > 0.05);

[0031] Figure 12Results of RT-qPCR detection of the effect of RPLys on the relative expression level of CAT1 mRNA in lymphocytes of each dairy cow in each experimental group; all were compared with the data on the 0th day of feeding, * indicates significant difference (p<0.05), ** indicates extremely significant difference (p<0.01), *** indicates extremely significant difference (p<0.001);

[0032] Figure 13 Results of q-PCR detection of the effect of RPLys on the proviral load of BLV in dairy cow lymphocytes; the proviral loads on the 10th, 20th, and 30th days were all compared with the data on the 0th day of feeding, and the proviral load on the 60th day was compared with that on the 30th day, * indicates significant difference (p<0.05), ** indicates extremely significant difference (p<0.01), *** indicates extremely significant difference (p<0.001), ns indicates no significant difference (p>0.05);

[0033] Figure 14 Results of digital PCR detection of the effect of RPLys on the proviral load of BLV in dairy cow lymphocytes. Detailed implementation manners

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in combination with specific implementation manners and the accompanying drawings of the specification. It should be noted that the embodiments mentioned below are only applicable to explaining the present invention, but not for limiting the scope of the present invention. The embodiments mentioned below are only a part of the embodiments of the present invention rather than all embodiments. In the art, for other technical personnel who have not made creative efforts, the embodiments obtained by them are protected by the present invention.

[0035] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified, and the materials, reagents, culture media and instruments used are all conventional materials, reagents, culture media and instruments in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels. The experimental operations involved in the present invention are all conventional experimental operations in the art or can be carried out in accordance with the product specifications of the corresponding reagents.

[0036] The experimental animals selected in the present invention: Holstein dairy cows randomly selected from a large-scale intensive ranch in Daqing City with similar ages, parities, etc.

[0037] The main reagents used in the present invention are as follows:

[0038] PrimeScript TM RT reagent Kit with gDNA Eraser (product number RR047A), TB GreenPremix Ex Taq TM(Product number: RR420A) was purchased from Baori Biotechnology Co., Ltd.; BCA Protein Concentration Assay Kit (Product number: P0010), Goat Anti-Mouse (Product number: A428), and Goat Anti-Rabbit (Product number: A423) were purchased from Beyotime Biotechnology Co., Ltd.; Bovine Peripheral Blood Lymphocyte Isolation Kit (Product number: P8640) and Red Blood Cell Lysis Buffer (Product number: R1010) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Tris (Product number: 1115) was purchased from Guangzhou Saiguo Biotechnology Co., Ltd.; Anti-CAT1 Antibody (Product number: ab37588) was purchased from Abcam Trading Co., Ltd.; 1×PBS Buffer (Product number: BL310A) and DEPC Water (RNase-free Water) (Product number: 700857) were purchased from Biosharp Life Sciences; Blood / Cell / Tissue Genomic DNA Extraction Kit (Product number: DP304) was purchased from Tiangen Biochemical Technology Co., Ltd.; RPLys was purchased from Beijing Feidi Feed Technology Co., Ltd.

[0039] The data processing and analysis methods used in this invention are as follows:

[0040] GraphPad Prism 8.0 software was used for statistical analysis and plotting of BLV proviral load, etc. ImageJ software was used for gray-scale analysis of the expression level of CAT1. The relative gene expression was analyzed using the 2 -ΔΔCt -ΔΔCt method. Each sample was measured at least 3 times, with 4 replicates each time. After sorting, GraphPad Prism 8.0 was used for analysis and plotting. Different data analysis methods were adopted according to the purpose of the obtained data, such as one-way ANOVA, etc. When P < 0.05, it indicates a significant difference between the data; when P < 0.01 or P < 0.001, it indicates an extremely significant difference between the data, with statistical significance.

[0041] Example 1: In vitro experiment to detect the proviral load in lymphocytes after BLV infects lymphocytes treated with different concentrations of Lys

[0042] 1. Screening of BLV-negative and positive cows (cows with high proviral load and cows with low proviral load)

[0043] (1) Detection of BLV-positive cows using ELISA kit

[0044] The collected whole blood of dairy cows was placed in a vacuum blood collection tube containing a coagulant; centrifuged at room temperature at 3000 rpm for 12 min, and the separated serum was placed in an EP tube and stored in a -80°C refrigerator; the bovine leukemia blocking antibody detection kit was used and operated according to the instructions.

[0045] (2) Extraction of whole blood DNA

[0046] Collect the whole blood of dairy cows. Take 300 μL and put it into an EP tube. Add 900 μL of red blood cell lysate, invert and mix well, and lyse at room temperature for 5 min; at room temperature, centrifuge at 1000 rpm for 1 min, discard the supernatant, and the white blood cell precipitate at the bottom can be seen. The remaining part is operated according to the instructions of the blood / cell / tissue genomic DNA extraction kit.

[0047] (3) Use absolute fluorescence quantitative PCR to detect the proviral load of BLV

[0048] The primers used in this detection method are BLV-F (nucleotide sequence as shown in SEQ ID NO.1: 5'-CCTCAATTCCCTTTAAACTA-3') and BLV-R (nucleotide sequence as shown in SEQ ID NO.2: 5'-GTACCGGGAAGACTGGATTA-3'), and the size of the pre-amplified fragment is 125 bp. The template used is a plasmid containing the full-length gene of BLV (pBLV344H, publicly available in the following literature: Li Siyan, He Sirui, Wang Yong, etc. BLV rescue and construction of its growth curve in dairy cow mammary epithelial cells [J]. Heilongjiang Animal Husbandry and Veterinary Medicine, 2021, (17): 17-20+144. DOI: 10.13881 / j.cnki.hljxmsy.2021.01.0250.), and this plasmid was kindly provided by Professor Luc Willems of the University of Liège, Belgium. The detected plasmid concentration is 252 ng / μL, and the copy number of the plasmid is calculated according to the formula to be 1.84×10 10 copies / μL. Dilute the plasmid by 10-fold gradient and make a standard curve (PCR reaction system: TB Green Premix Ex Taq TM 12.5 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, template DNA 2 μL, dH2O 9.5 μL; PCR reaction program is as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, 40 cycles), according to the R-square value of the standard curve, finally select the plasmid concentration range of 1.84×10 9 ~1.84×10 1 copies / μL (copy number calculation formula: [(6.02×10 23 )×(ng / μL×10 -9 )] / (DNALength×660)).

[0049] Referring to the method for detecting the proviral load of BLV in the OIE laboratory, we transformed and amplified the obtained pBLV344H plasmid and performed 10-fold gradient dilution, and finally selected the concentration range of 1.84×10 9 ~1.84×10 1copies / μL. Using this as a template, the proviral load in the sample was quantified, and the virus copy number was calculated. According to the research recommendations, a virus copy number > 1000 copies / 10 ng DNA was defined as high proviral load (HPVL), and otherwise as low proviral load (LPVL). Figure 1 is the standard curve of the template, and the correlation coefficient R 2 > 0.99, indicating a good linear relationship between the CT value and the concentration of the BLV plasmid standard, which can be used for subsequent experiments.

[0050] The isolated serum samples were detected using a bovine leukemia blocking antibody detection kit and analyzed by xChek software. Among the 70 samples, 18 samples showed serological positivity, with a positive rate of 25.71%. Then, the proviral load of these 18 samples was detected using absolute quantitative PCR. Among the 18 samples, 8 dairy cows had a high virus load, accounting for 44% of the positive cows (see Table 1).

[0051] Table 1 Screening results of BLV proviral load in BLV-positive cows

[0052]

[0053] 2. Isolation, culture, and identification of peripheral blood lymphocytes from healthy dairy cows

[0054] (1) Isolation of peripheral blood lymphocytes

[0055] ① The whole blood of the healthy dairy cows obtained by the above screening was collected using a vacuum blood collection tube containing an anticoagulant, and the subsequent steps were strictly carried out under aseptic conditions;

[0056] ② Add 4 mL of lymphocyte separation solution to a 15 mL centrifuge tube, and then add 4 mL of the collected dairy cow whole blood (note: slowly inject the blood along the side wall to keep the interface of the two liquid surfaces clear);

[0057] ③ At room temperature, centrifuge at 1000 × g for 30 min using a horizontal rotor;

[0058] ④ After centrifugation, obvious stratification will occur. Carefully aspirate the middle white lymphocyte layer into a new 15 mL centrifuge tube, add 9 mL of cell washing solution, and centrifuge at 250 × g for 10 min at room temperature;

[0059] ⑤ Discard the supernatant, observe the color of the cell pellet. If there are red blood cells mixed in, add 3 mL of red blood cell lysate, lyse at room temperature for 3 min, and centrifuge at 250 × g for 8 min;

[0060] ⑥ Discard the supernatant, wash again, centrifuge at 250 × g for 8 min, and the obtained cell pellet is the lymphocyte pellet, which is reserved for use.

[0061] (2) Counting of lymphocytes

[0062] Resuspend the isolated lymphocytes with 1 mL of PBS and mix well. Aspirate 10 μL of the cell suspension and 10 μL of trypan blue, and pipette them up and down to mix. Withdraw 10 μL of the mixture and drop it onto a cell counting plate, then count with a cell counter. Repeat three times.

[0063] (3) Cultivation of lymphocytes

[0064] ① First, put supplies such as 25 cm 2 cell culture flasks, pipettors, electric pipette aids, pipettes, etc. into the laminar flow hood and turn on the ultraviolet sterilization for more than 30 min;

[0065] ② Preheat the 1640 medium in a 37 °C water bath;

[0066] ③ Prepare the complete medium (5% fetal bovine serum, 1% double antibody);

[0067] ④ Aspirate 4 mL of the complete medium into a 25 cm 2 cell culture flask, then resuspend the lymphocyte pellet with 1 mL of the complete medium, adjust the cell density to 2×10 6 cells / mL, and finally mix the two;

[0068] ⑤ Add concanavalin A to make the final concentration of concanavalin A 10 μg / mL;

[0069] ⑥ Incubate in a 37 °C, 5% CO2 cell culture incubator.

[0070] Collect the whole blood of healthy dairy cows, isolate lymphocytes and culture them in vitro. First, perform a viable cell count on the isolated lymphocytes. Figure 2 As shown in A of 6 / mL; Figure 2 As shown in B of

[0071] 3. Detect the effect of Lys on the proliferation of lymphocytes cultured in vitro

[0072] Detect cell proliferation by CCK8 method:

[0073] ① After culturing lymphocytes for 24 h, culture them with an induction medium without fetal bovine serum for 8 h;

[0074] ② Place the lymphocytes in 1640 medium without fetal bovine serum at 8 different Lys concentrations respectively for culture, plate in a 96-well plate, 200 μL per well, containing 2×105 Lymphocytes. On each plate, a solvent control group and 1640 medium without fetal bovine serum containing 8 different Lys concentrations (Lys: 0.27, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 mmol / L) were set up. Each group had 4 replicates and was placed in a cell culture incubator for continued culture. The culture times were 24, 36, 48, 60, and 72 h respectively;

[0075] ③ One hour before the end of the culture, 20 μL of CCK8 solution was added to each culture well, protected from light, and then continued to be cultured in a 37 °C, 5% CO2 cell culture incubator (note that when adding the CCK8 solution, it should be protected from light. After adding, wrap the 96-well plate with tin foil and then put it into the cell culture incubator);

[0076] ④ The OD value of each well at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0077] The calculation formula for cell survival rate is: Cell survival rate = [(absorbance of test well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100%.

[0078] The effect of different concentrations of Lys on the proliferation activity of lymphocytes cultured in vitro can be represented by the cell survival rate. As shown in Table 2: Lys at 0.4 - 6.4 mmol / L had a significant promoting effect on the proliferation of lymphocytes cultured in vitro (P < 0.05), the promoting effect of Lys at 12.8 mmol / L was not obvious, and Lys at 25.6 mmol / L had a significant inhibitory effect on the proliferation of lymphocytes cultured in vitro (P < 0.05). Moreover, the promoting effect on the proliferation of lymphocytes cultured in vitro was the most obvious at a culture time of 48 h (P < 0.05). Finally, Lys concentrations of 0.27 mmol / L (Ctrl group: the concentration of Lys contained in normal 1640 medium), 3.2 mmol / L (low Lys concentration group), and 12.8 mmol / L (high Lys concentration group) were selected, and the stimulation time was 48 h for subsequent cell experiments.

[0079] Table 2 Results of the effects of different concentrations of lysine and different treatment times on the proliferation of lymphocytes cultured in vitro

[0080]

[0081] Note: Different lowercase letters in the table indicate significant differences in lymphocyte survival rates at the same time under different lysine concentrations (P < 0.05); different capital letters in the table indicate significant differences in lymphocyte survival rates at the same lysine concentration at different times (P < 0.05)

[0082] 4. Detect the effect of Lys on the relative expression levels of CAT1 protein and CAT1 mRNA in lymphocytes

[0083] Detect the effect of Lys on the expression level of CATI protein in lymphocytes

[0084] (1) Isolation of peripheral blood lymphocytes

[0085] Collect the whole blood of healthy dairy cows again and isolate lymphocytes (since lymphocytes cannot survive for a long time in vitro, fresh blood collection and isolation are required for each experiment). The specific method is referred to the above-mentioned isolation method of peripheral blood lymphocytes.

[0086] (2) Treatment of lymphocytes

[0087] The isolated lymphocytes were first cultured in complete medium for 24 h, then transferred to induction medium (without fetal bovine serum) for starvation culture for 8 h, and then cultured under the three selected Lys concentrations for 24 h for subsequent experiments.

[0088] (3) Extraction of total cellular protein

[0089] ① Prepare the lysis buffer (RIPA:PMSF = 100:1, RIPA:protease inhibitor = RIPA:phosphatase inhibitor = 50:1);

[0090] ② Add an appropriate amount of the prepared lysis buffer to the lymphocyte pellet and lyse on ice for 3 min;

[0091] ③ Centrifuge at 12000 rpm for 15 min at 4 °C, take the supernatant and transfer it to a pre-cooled EP tube, measure the protein concentration, and store at -80 °C.

[0092] (4) Determination of protein concentration

[0093] ① Operate according to the instructions of the BCA protein concentration assay kit;

[0094] ② Plot the standard curve, calculate the sample concentration, adjust the protein concentration to the same level with RIPA lysis buffer and 5×Buffer, denature the protein by boiling in water for 10 min, and store at -20 °C.

[0095] (5) Detection of protein level

[0096] ① Prepare the SDS-PAGE gel: Prepare a suitable separating gel according to the protein molecular weight. The concentration of the separating gel prepared in this experiment is 10%. The method is shown in Table 3. After preparation, take 4.5 mL and add it to the gel plate, add isopropanol to press the gel, and let it stand at room temperature for 1 h. Pour off the upper isopropanol, prepare the stacking gel, the method is shown in Table 3. After preparation, add it on the surface of the separating gel, insert the comb, and let it stand for 45 min for later use;

[0097] Table 3 SDS-PAGE Gel Recipe

[0098]

[0099] ② Loading and Electrophoresis: Remove the comb, and add the samples into the wells in sequence. The loading volume for the protein well is 30 μg, and the loading volume for the Mark well is 3 μL. Set the initial voltage to 80 V for 30 min, then change the voltage to 120 V. Stop the electrophoresis when the bromophenol blue indicator band reaches the bottom of the separating gel;

[0100] ③ Transfer: Use the rapid transfer solution. According to the size of the protein molecules in this experiment, the current condition is 400 mA for 30 min to complete the transfer;

[0101] ④ Blocking: Rinse the transferred PVDF membrane once in TBS-T for 2 min, then place it in 5% skim milk for blocking for 2 h;

[0102] ⑤ Primary Antibody Incubation: Rinse twice with TBS-T for 3 min each time, and incubate with the primary antibody (diluted with TBS-T) overnight (place it on a shaker at 4°C for about 10 h);

[0103] ⑥ Membrane Washing: First rinse three times with TBS-T, shaking rapidly for 5 min each time, then rinse three more times, shaking rapidly for 10 min each time;

[0104] ⑦ Secondary Antibody Incubation: Select the corresponding secondary antibody according to the species of the primary antibody, and incubate on a shaker at room temperature for 1.5 h;

[0105] ⑧ Membrane Washing: First rinse three times with TBS-T, shaking rapidly for 5 min each time, then rinse three more times, shaking rapidly for 10 min each time;

[0106] ⑨ ECL Development: Evenly drip the immobilon western luminescent agent onto the PVDF membrane band, observe the results with an Amersham Imager 600, and perform gray-scale analysis using ImageJ-win 64 software.

[0107] Detect the Effect of Lys on the Relative Expression Level of CAT1 mRNA in Lymphocytes

[0108] (1) Extraction of Total RNA from Lymphocytes

[0109] ① The method of treating lymphocytes is the same as that for detecting the CAT1 protein expression level;

[0110] ② Extract the total RNA from lymphocytes according to the conventional TRIzol method in the laboratory.

[0111] (2) cDNA Synthesis

[0112] ① Using the PrimeScriptTM RT reagent Kit with gDNA Eraser reverse transcription kit, prepare the reaction solution on an ice box. First, remove genomic DNA. The reaction system is as follows: 1.0 μL of gDNA Eraser, 2.0 μL of 5×gDNA Eraser Buffer, ≤1 μg of total RNA, and make up to 10 μL with RNase Free ddH2O;

[0113] ② Place the reaction system in a 42°C water bath and react for 2 min;

[0114] ③ Configure the reverse transcription reagent on an ice box. The reverse transcription system is as follows: 10.0 μL of the reaction solution obtained in the previous step, 4.0 μL of RT Primer Mix, 1.0 μL of RNase Free dH2O, 24.0 μL of 5×PrimeScript Buffer, and 11.0 μL of PrimeScript RT Enzyme Mix;

[0115] ④ Place the RNase-free centrifuge tube containing the reaction solution in a 37°C water bath. After 15 min, transfer it to an 85°C water bath for 5 s to obtain cDNA, and store it at -20°C.

[0116] (3) Detect the relative expression level of CAT1 mRNA

[0117] The fluorescent quantitative PCR primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 4. The RT-qPCR amplification system is as follows: 12.5 μL of TB Green Premix Ex TaqTM, 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 2.0 μL of cDNA, and 9.5 μL of dH2O; The RT-qPCR amplification program is as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, 95°C for 10 s, 40 cycles; 60°C for 5 s.

[0118] Table 4 Primer sequence information for real-time fluorescence quantitative PCR

[0119]

[0120]

[0121] The isolated healthy dairy cow lymphocytes were cultured for 24 h at Lys concentrations of 0.27 mmol / L (Ctrl group: the Lys concentration contained in normal 1640 medium), 3.2 mmol / L (low Lys concentration group), and 12.8 mmol / L (high Lys concentration group) to observe the effect of Lys concentration on the expression levels of CAT1 protein and mRNA in the cells. The results showed that: compared with the Ctrl group, the expression levels of CAT1 protein and mRNA in the cells of the low Lys concentration group were significantly increased (P<0.05), and the expression levels of CAT1 protein and mRNA in the cells of the high Lys concentration group were extremely significantly increased (P<0.001) (see Figure 3 ). From the above results, it can be seen that the expression level of CAT1 can be regulated by the Lys concentration, and with the increase of the Lys concentration, the expression of CAT1 also increases.

[0122] 5. Acquisition of BLV and detection of BLV proviral load

[0123] (1) Culture of BL3.1 cells

[0124] BL3.1 cells are bovine B lymphocytes that can continuously secrete bovine leukemia virus particles and are purchased from the ATCC cell bank.

[0125] Resuscitation of BL3.1 cells:

[0126] Turn on the water bath in advance, set it to 37 °C, and put supplies such as 25 cm 2 cell culture flasks, pipettes, electric pipette aids, pipettes, etc. into the laminar flow hood and turn on the ultraviolet sterilization for more than 30 min. Take out the cryopreserved BL3.1 cells from liquid nitrogen, quickly put them into a 37 °C water bath, transfer them into the laminar flow hood when only a small piece of ice remains, melt the remaining ice with the residual temperature, place them in a 15 mL centrifuge tube, slowly and then quickly add 5 mL of 1640 complete medium to the 15 mL centrifuge tube, centrifuge at 1000 rpm for 4 min, discard the supernatant, resuspend the cell pellet, and inoculate it into a 25 cm 2 cell culture flask, and culture it in a 37 °C, 5% CO2 cell culture incubator. Observe the status the next day.

[0127] Passage of BL3.1 cells:

[0128] Use a pipette to disperse the aggregated cell clumps and inoculate them into a 75 cm 2 cell culture flask, add 5 mL of 1640 complete medium, and culture it in the cell culture incubator for two days. Observe the cell status during this period and gently shake the culture flask. On the third day, disperse the clumps and add 10 mL of 1640 complete medium, and culture again.

[0129] Cryopreservation of BL3.1 cells:

[0130] Prepare the cryopreservation solution in advance by mixing DMSO and fetal bovine serum at a ratio of 1:9, and let it stand for about 3 minutes after preparation. Take out the cultured BL3.1 cells from the incubator, centrifuge at 1000 rpm for 4 minutes, discard the supernatant, wash the cells with PBS, centrifuge at 1000 rpm for 4 minutes. For the cells in a 25 cm 2 cell culture flask, resuspend them with 1 mL of the cryopreservation solution, place them in a cryotube, make good marks, put the cryotube into a cell programmable cooling box, then put it into an -80 °C refrigerator for 24 hours, and finally transfer it to liquid nitrogen for long-term storage.

[0131] (2) Observation of virus particle production in BL3.1 cells by transmission electron microscopy

[0132] To determine whether BL3.1 cells can produce BLV particles, it was identified by electron microscopy observation. This part of the experiment was entrusted to the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences to complete.

[0133] The results showed that virus-like particles with a diameter of 80 - 120 nm could be seen at the edge of the lymphocyte membrane, and the number of virus particles was relatively large at the edge of the cell membrane near the Golgi apparatus.

[0134] (3) Determination of the BLV infection time

[0135] ① Take out the BL3.1 cells cultured for a sufficient time from the cell incubator, centrifuge at 2000 rpm for 20 minutes, take the supernatant, centrifuge at 10000 rpm for 20 minutes, take the supernatant, centrifuge at 100000 rpm for 60 minutes, discard the supernatant, and resuspend the precipitate with the basic medium. This is the virus solution;

[0136] ② Add 1 mL of the virus solution to the healthy dairy cow lymphocytes being cultured, and collect the RNA and DNA of the lymphocytes at 24 h and 48 h after infection respectively. The method is the same as above;

[0137] ③ Perform ordinary PCR on the samples to determine the infection time. The primers for PCR are BLV-F (SEQ ID NO.1) and BLV-R (SEQ ID NO.2). The ordinary PCR amplification system for the target gene is as follows: upstream primer 0.5 μL, downstream primer 0.5 μL, cDNA 2 μL, Taq Master Mix 12.5 μL, RNase-Free Water 9.5 μL. The ordinary PCR amplification program for the target gene is as follows: 94 °C for 5 minutes; 94 °C for 30 s, 60 °C for 30 s, 72 °C for 10 s, 40 cycles; 72 °C for 10 minutes.

[0138] ④ Mix the PCR product with 10×M Buffer and perform 2% agarose gel electrophoresis detection.

[0139] Differentially centrifuged virus solution was extracted from BL3.1 cells and used to infect healthy dairy cow lymphocytes. After 24 h, DNA and mRNA were extracted from the cells. The mRNA was reverse transcribed into cDNA and subjected to nucleic acid electrophoresis. The results showed that the virus had completely infected healthy dairy cow lymphocytes after 24 h (see Figure 4 ).

[0140] (4) BLV infection of dairy cow lymphocytes

[0141] The BLV infection time was determined by nucleic acid electrophoresis and analyzed in combination with the time of the effect of Lys on lymphocyte proliferation to obtain an optimal time for co-treatment to treat the cells (for example, BLV could infect the cells in 24 h, and the promoting effect of Lys on cell proliferation activity was the strongest at 48 h. Then the optimal time for co-treatment was to first treat lymphocytes with different concentrations of Lys for 24 h and then add BLV to stimulate for 24 h respectively). After the treatment, DNA and RNA were collected.

[0142] (5) Detection of the change in BLV proviral load using digital PCR

[0143] ① Prepare the reaction solution on an ice box (10 μL of qPCR mix, 1 μL each of upstream and downstream primers (10 μM), 1 μL of template DNA, 11 μL of ddH2O, and 1 μL of Alexa647 reference fluorescence). Add all the reagents to a centrifuge tube and centrifuge briefly to collect all the reagents at the bottom of the tube and remove air bubbles;

[0144] ② Load the sample to be tested: Open the lid of the PCR chip, inject the prepared 25 μL of PCR reaction solution into the sample loading hole of the chip, and cover the chip with the special PCR sealing hole cover. Note that the pipette tip should not contact the internal oil phase, and no air bubbles should be generated throughout the process;

[0145] ③ At this time, transfer the sealed chip to the Naica Geode microdroplet generation and amplification system, adjust the air pressure between 1100 - 1200 bar, open the upper cover of the machine, place the chip in the heating module, and start the program;

[0146] ④ After the program ends, take out the chip for data collection. Use the computer controlling the Naica Prism 3, and use the Crystal Reader software to call the pre-set scanning program to scan the chip parameters. After the scanning ends, the result pictures can be directly exported according to the requirements.

[0147] After culturing healthy dairy cow lymphocytes in the Ctrl group, low Lys concentration group, and high Lys concentration group for 24 h, 1 mL of virus was added to stimulate for 24 h, DNA samples were collected, and digital PCR technology was used to detect the BLV proviral load. The results showed that compared with the Ctrl group, the BLV proviral loads in the low Lys concentration group and high Lys concentration group increased extremely significantly (P<0.001). Compared with the low Lys concentration group, the BLV proviral load in the high Lys concentration group decreased extremely significantly (P<0.001) (see Figure 5 ).

[0148] According to the results of the effect of Lys on the BLV proviral load in cultured dairy cow lymphocytes in vitro, compared with the Ctrl group, the BLV proviral loads in the low Lys concentration group and high Lys concentration group increased extremely significantly. This is because Lys promoted the expression of CAT1, resulting in an increase in the BLV proviral load. However, compared with the low Lys concentration group, the BLV proviral load in the high Lys concentration group decreased significantly. Normally, within a certain concentration range, the expression of CAT1 is dose-dependent on the Lys concentration. The expression of CAT1 in the high Lys concentration group increased, and the proviral load should have increased, but the result was the opposite. Therefore, we speculated that high-dose Lys competitively binds to CAT1 with BLV, resulting in a decrease in the virus load. Subsequently, based on this result, we conducted in vivo experiments to further determine whether Lys really has a competitive inhibitory effect on BLV entry into cells.

[0149] Example 2: In vivo experiment to detect the effect of RPLys on the BLV proviral load in lymphocytes of BLV-positive dairy cows

[0150] According to the previous screening results, 21 Holstein dairy cows with similar ages and body conditions were selected, including 7 HPVL cows, 7 LPVL cows, and 7 BLV-negative cows for subsequent experiments. The screening results of the proviral loads of the 21 selected dairy cows are shown in Table 5.

[0151] Table 5 Screening results of BLV proviral load

[0152]

[0153]

[0154] 1. Index detection before feeding RPLys

[0155] (1) Detection of amino acid concentration in lymphocytes

[0156] Before the start of feeding, the amino acid concentrations in lymphocytes of BLV-negative, LPVL, and HPVL dairy cows were detected to preliminarily determine whether cationic amino acids competitively bind to CAT1. The specific method is as follows:

[0157] ① Sample preparation: Collect whole blood from experimental animals to isolate lymphocytes. Count the isolated lymphocytes on a cell counter (the cell amount of each sample should reach at least 10 7 ), record the data. After washing the cells twice, add 100 μL of ultrapure water to each to make the cells swell and burst, releasing free amino acids;

[0158] ② Submit for inspection (entrust the Biological Laboratory of Peking University to detect the samples using an automatic amino acid analyzer);

[0159] ③ Use an automatic amino acid analyzer to measure the content of various free amino acids in the sample. First, remove the fat impurities;

[0160] ④ Transport the processed sample to the chromatographic column through an autoinjector for separation. Use buffer solutions with different pH values and ionic concentrations to elute each amino acid component in turn, then mix with ninhydrin reagent in another flow path one by one, and then flow together into a spiral reaction tube for color reaction at a certain temperature (usually 115 - 120 °C). The generated purple substance is colorimetrically determined at a wavelength of 570 nm, and the generated yellow compound is colorimetrically determined at a wavelength of 440 nm. Finally, accurate data are given by an automatic computer.

[0161] The detection and analysis of the amino acid content in lymphocytes of 21 selected dairy cows showed that: compared with BLV-negative cows, the contents of Lys, His, and Arg in lymphocytes of BLV-positive cows showed an upward trend, but there was no significance (P > 0.05). Compared with the LPVL group, the contents of Lys and His in lymphocytes of the HPVL group showed a slight upward trend, without significance (P > 0.05), and the content of Arg showed a slight downward trend, without significance (P > 0.05) (see Figure 6 ). Therefore, to know whether cationic amino acids have the effect of competitively binding to CAT1, further experiments are needed.

[0162] (2) Detection of the relative expression level of CAT1 protein in lymphocytes of BLV-negative and positive dairy cows

[0163] To observe the effect of BLV on CAT1 protein, we detected the relative expression level of CAT1 protein in lymphocytes of BLV-negative and positive dairy cows using Western blot. The method is detailed in Example 1.

[0164] Before the start of feeding, the expression levels of CAT1 protein in lymphocytes of BLV-negative cows, LPVL cows, and HPVL cows were detected. The results showed that compared with BLV-negative cows, the expression level of CAT1 protein in cells of BLV-positive cows had an upward trend but was not significant (P>0.05) (see Figure 7 ).

[0165] 2. Index detection after feeding RPLys

[0166] Six Holstein cows with similar ages and lactation status were selected from HPVL cows and LPVL cows as experimental subjects, and were divided into HPVL group (n = 4), LPVL group (n = 4), and Ctrl group (n = 2 + 2, 2 HPVL cows + 2 LPVL cows). The HPV group and LPVL group were fed 50 g of RPLys every day for 30 days, and the Ctrl group had a normal diet.

[0167] The day before feeding was defined as day 0. Blood samples were collected on days 0, 10, 20, 30, 45, 60, and 90 respectively. After collection, DNA was extracted, lymphocytes were separated, total cellular protein and RNA were extracted for detection of relevant data.

[0168] After feeding RPLys, the relative expression levels of CAT1 protein and mRNA in lymphocytes of cows were first detected; subsequently, the changes in BLV proviral load were detected by absolute fluorescence quantitative PCR and digital PCR respectively. The detailed steps of the above experiments can be seen in Example 1.

[0169] (1) Effect of RPLys on the expression level of CAT1 protein in lymphocytes of cows

[0170] After HPVL group and LPVL group were fed RPLys, the expression levels of CAT1 protein in lymphocytes increased to varying degrees, while the expression level of CAT1 protein in lymphocytes of Ctrl group did not change significantly. Comprehensive analysis showed that: in the HPVL group, compared with day 0, on days 20 and 30 after feeding RPLys, the expression level of CAT1 protein in lymphocytes increased extremely significantly (p<0.001), and compared with day 20, on day 30, the expression level of CAT1 protein in lymphocytes increased significantly (p<0.05); in the LPVL group, compared with day 0, on days 20 and 30 after feeding RPLys, the expression level of CAT1 protein in lymphocytes increased significantly (p<0.05, p<0.001), and compared with day 20, on day 30, the expression level of CAT1 protein in lymphocytes did not change significantly (p>0.05); in the Ctrl group, compared with day 0, on days 20 and 30 after normal diet, the expression level of CAT1 protein in lymphocytes did not change significantly, and there was no statistical difference (p>0.05) (see Figure 8, Figure 9 and Figure 10 )。

[0171] (2) Effect of PRLys on the relative expression level of CAT1 mRNA in dairy cow lymphocytes

[0172] The relative expression level of CAT1 mRNA in dairy cow lymphocytes was detected by RT-qPCR. The results showed that in the HPVL group and the LPVL group, compared with the 0th day of feeding RPLys, the expression level of CAT1 mRNA in dairy cow lymphocytes was extremely significantly increased on the 30th day (p < 0.001); in the Ctrl group, compared with the 0th day of feeding RPLys, there was no significant change in the expression level of CAT1 mRNA in dairy cow lymphocytes on the 30th day, and there was no statistical difference (p > 0.05) (see Figure 11 and Figure 12 )。

[0173] (3) Detection of changes in BLV proviral load in dairy cows by absolute fluorescence quantitative PCR

[0174] The changes in proviral load in each group of dairy cows could be divided into two stages: the stage of feeding RPLys for the first 30 days and the tracking stage for the next 30 days. The results showed that in the HPVL group and the LPVL group, from 0 to 10 days of feeding RPLys, the BLV proviral load showed an obvious upward trend, and the difference was significant compared with the 0th day (P < 0.05). From 10 to 20 days, three cows showed an upward trend and three cows showed a downward trend. From 20 to 30 days, one cow showed an upward trend and five cows showed a downward trend. From 10 to 30 days, the overall trend was downward, and the proviral load on the 30th day was significantly lower than that on the 20th day. From 0 to 30 days, the proviral load of the Ctrl group showed a fluctuating upward trend. From 30 to 60 days, the proviral load of the three groups of experimental cows showed an upward trend, and the proviral load on the 60th day was significantly increased compared with the 30th day (P < 0.05) (see Figure 13 )。

[0175] (4) Detection of changes in BLV proviral load in dairy cows by digital PCR

[0176] This part is to verify the results of viral load obtained by absolute quantitative PCR using digital PCR. Digital PCR is an absolute quantification technique for nucleic acid molecules. Compared with qPCR, digital PCR can directly count the number of DNA molecules and is an absolute quantification of the starting sample with relatively high accuracy. The results show that in the HPVL group and the LPVL group, during the 0 - 20 days of feeding RPLys, the proviral load of BLV showed an obvious upward trend, indicating that Lys affected the expression of CAT1, thus leading to a rapid increase in the proviral load of BLV in the short term; during the 20 - 30 days, the proviral load of BLV showed an obvious downward trend. This may be because when the amount of Lys given was constant, the expression level of CAT1 did not change significantly, and Lys played a role in competitively binding to CAT1 with BLV, resulting in a decrease in the proviral load. During the 0 - 30 days, the change in the proviral load of BLV in the experimental group was relatively large with large fluctuations. Relatively speaking, the Ctrl group showed a steadily increasing trend, indicating that RPLys had a certain impact on the viral load. During the 30 - 90 days, the proviral load of the three groups of experimental cows all showed an upward trend (see Figure 14 ), which may be because although the feeding of RPLys was stopped, the expression of CAT1 did not return to the previous level immediately and was still in a state of increased expression in the short term, so it led to a rapid increase in the proviral load. Such results suggest that Lys can affect the proviral load of BLV in dairy cow lymphocytes. Lys affects the BLV load by influencing the expression of CAT1. Therefore, for BLV-negative cows, excessive supplementation of Lys in vitro has no preventive effect on BLV, but instead increases the susceptibility to BLV.

[0177] In summary, feeding rumen-protected lysine at a dose of 50 g per day for 30 days can slow down the upward trend of the proviral load in BLV-positive cows.

[0178] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of lysine in the preparation of a medicament for slowing down the increase in the proviral load of BLV in BLV-positive bovine lymphocytes.

2. The application according to claim 1, characterized in that, The lysine is rumen-protected lysine.

3. The application according to claim 2, wherein The administration mode of the medicament is oral administration.

4. The application according to claim 3, characterized in that The dosage of rumen-protected lysine in the medicament is 50 g / d.

5. The application according to claim 1, characterized in that The medicament further comprises a pharmaceutically acceptable carrier.

6. Use of lysine in the preparation of a medicament for relieving bovine leukemia.

7. The application according to claim 1, wherein The lysine is rumen-protected lysine.

8. The application according to claim 1, characterized in that, The administration mode of the medicament is oral administration.

9. The application according to claim 1, characterized in that, The dosage of rumen-protected lysine in the medicament is 50 g / d.

10. The application according to claim 1, wherein The medicament further comprises a pharmaceutically acceptable carrier.

Images