Application of a recombinant chicken thymosin in the preparation of an immune enhancer for chickens

Abstract

The present invention provides an application of recombinant chicken thymopeptide in the preparation of chicken immune enhancers, belonging to the field of veterinary technology. In the present invention, chicken thymosin α1, chicken thymosin α1 mutant and double-copy tandem protein of chicken thymosin α1 mutant are expressed by genetically engineered recombinant Escherichia coli. The action time of recombinant chicken thymopeptide in chickens is increased through two ways: mutating the positively charged amino acid K into the negatively charged amino acid D and double-copy tandem expression. Seven weeks after the injection of thymopeptide, the SI value of the chicken thymosin α1 group has dropped to 1.08, approaching the control group level, the SI value of the chicken thymosin α1 mutant group has dropped to 1.23, while the SI value of the double-copy chicken thymosin α1 mutant group still maintains a relatively high level of 1.43. It can be seen that the double-copy chicken thymosin α1 mutant has a more persistent effect on regulating the immune function of chickens than chicken thymosin α1 expressed by the natural sequence, and is a long-acting thymopeptide that can be used as an immune enhancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary medicine, and particularly relates to the application of recombinant chicken thymosin in the preparation of chicken immune enhancers. Background Art

[0002] In recent years, the large-scale prevalence of immunosuppressive diseases has become one of the main threats to the livestock and poultry breeding industry. Immunosuppressive diseases lead to low immunity in livestock and poultry, making them vulnerable to multiple infectious diseases. At the same time, they also cause poor immune responses to vaccines in livestock and poultry, resulting in immune failure and the widespread prevalence of infectious diseases. Immunomodulatory factors such as thymosin can activate the body's immune response and enhance the body's immunity, and are effective bioactive substances for improving immune suppression in the body. Thymosin can induce the differentiation and maturation of T cells, enhance the production of cytokines, and enhance the antibody response of B cells. The most important role of thymosin is to continuously induce all stages of the differentiation and development of T cells, maintain the body's immune balance, enhance the response of T cells to antigens, and thus improve the body's ability to resist diseases. Existing research results show that the main active component of thymosin is thymosin α1 composed of 28 amino acids, and it has been applied in human medicine.

[0003] Livestock and poultry have a well-developed thymus in their juvenile stage. As the body grows, the thymus gradually atrophies, and the immune regulatory role dominated by thymosin also drops linearly. Natural thymosin is extracted from the thymus of juvenile livestock and poultry. The insufficient thymus source and the complex extraction process result in the high cost of natural thymosin, which hinders the application of natural thymosin in livestock and poultry breeding. Through genetic engineering technology, the large-scale expression and production of thymosin can be realized, making it possible to popularize the use of genetically engineered thymosin in livestock and poultry breeding. However, the molecular weight of the expressed thymosin is less than 10 kD. The kidney filtration system is easy to remove substances smaller than the kidney filtration cut-off size and not bound to plasma proteins. Thymosin is easily cleared by the kidneys. The half-life of the genetically engineered drug Neulasta for injection in humans is only 1.6 hours, and it needs to be injected twice a week for 6 consecutive months. In addition to the molecular weight, the charge carried by the substance is also an important factor affecting kidney clearance. Since the basement membrane of the kidney is negatively charged, under the same conditions, cationic polypeptides are often cleared faster. Currently, both thymosin extracted from the thymus and thymosin obtained by expressing the natural thymosin sequence have the defects of being easily cleared by the kidneys due to their molecular weight being less than the kidney filtration cut-off size and having too short a physiological activity time in the body. Repeated injection of thymosin will increase the drug cost and labor cost, and at the same time will cause animal stress and reduce the production of livestock and poultry. Therefore, developing long-acting thymosin, prolonging the retention time of thymosin in the body plasma, and further improving the immune regulatory effect of thymosin on the body are urgent problems to be solved in the application of thymosin in livestock and poultry breeding. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of recombinant chicken thymosin in the preparation of chicken immune enhancers, belonging to the technical field of veterinary medicine.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] First, the present invention provides an application of a recombinant chicken thymopeptide in the preparation of a chicken immune enhancer, and the amino acid sequence of the recombinant chicken thymopeptide is SEQ ID NO.5.

[0007] Furthermore, the nucleotide sequence of the recombinant chicken thymopeptide is SEQ ID NO.6.

[0008] Furthermore, the preparation method of the recombinant chicken thymopeptide comprises the following steps:

[0009] (1) Pick a single colony of E.coli BL21 / pET28a-Tα1M2 and inoculate it into a 10 ml LB culture medium supplemented with kanamycin, and culture it at 37°C with shaking until the OD600nm reaches 0.6 - 0.8;

[0010] (2) Add 0.5 mol / L α-lactose to a final concentration of 0.03 mol / L and culture it at 37°C for another 6 h;

[0011] (3) Centrifuge at 12000 r / min for 10 min to collect the bacterial cells. Add PBS with a volume 10 times that of the bacterial cell weight to suspend the bacterial cells, and disrupt them by ultrasonic wave. Centrifuge at 4°C and 12000 r / min for 15 min to collect the supernatant;

[0012] (4) Filter the supernatant through a 0.22 μm filter, purify it with a nickel column, and store the purified protein at -20°C for standby.

[0013] Second, the present invention provides a chicken thymopeptide α1 mutant, and the amino acid sequence of the chicken thymopeptide α1 mutant is SEQ ID NO.3.

[0014] Furthermore, the nucleotide sequence of the chicken thymopeptide α1 mutant is SEQ ID NO.4.

[0015] Beneficial effects: The present invention conducts a comparative analysis on the amino acid sequences of human thymosin α1 and chicken thymosin α propeptide, and selects the corresponding amino acid sequences of both as the chicken thymosin α1 sequence for protein expression. To extend the half-life of recombinant chicken thymosin α1 in chickens, the amino acid sequence of chicken thymosin α1 is analyzed, and the positively charged amino acid K at position 21 is mutated into the negatively charged amino acid D to obtain the amino acid sequence of the chicken thymosin α1 mutant. To overcome the drawback that chicken thymosin α1 is easily cleared by the kidneys due to its molecular weight being smaller than the renal filtration cut-off size, a tandem sequence of two copies of the chicken thymosin α1 mutant is designed, and the two copies are connected by the SG flexible peptide, thereby designing the amino acid sequence for the expression of the tandem protein of the chicken thymosin α1 mutant. Recombinant Escherichia coli is used to express chicken thymosin α1, the chicken thymosin α1 mutant, and the tandem protein of the chicken thymosin α1 mutant. All three recombinant chicken thymosins have good biological activities, and the percentage of E rosettes is higher than 48.93%. Tandem expression prolongs the action time of recombinant chicken thymosin in chickens. Seven weeks after the injection of thymosin, the SI value of the chicken thymosin α1 group has decreased to 1.08, approaching the control group level, the SI value of the chicken thymosin α1 mutant group has decreased to 1.23, while the SI value of the double-copy chicken thymosin α1 mutant group still maintains a relatively high level of 1.43. It can be seen that the double-copy chicken thymosin α1 mutant has a more persistent effect on regulating the immune function of chickens than chicken thymosin α1 expressed by the natural sequence, and is a long-acting thymosin that can be used as an immune enhancer. Brief Description of the Drawings

[0016] Figure 1 Conduct a comparative analysis on the amino acid sequences of human thymosin α1 and chicken thymosin α propeptide;

[0017] Figure 2 Determine the activity of recombinant thymosin by the E rosette test;

[0018] Figure 3 Detect the immune enhancement activity of recombinant chicken thymosin in chickens. Detailed Embodiments

[0019] Example 1: Expression of Chicken Thymosin α1 Protein

[0020] (1) Comparative analysis of the mature peptide sequence of chicken thymosin α1 It is known that the main active component of human thymosin is thymosin α1 composed of 28 amino acids, and its amino acid sequence accession number is PDB: 2L9I_A. There is little research on chicken thymosin α, and only the propeptide sequence of chicken thymosin α, XP_040534648.1, is registered in GenBank, but the amino acid sequence of mature chicken thymosin α is not available. Conduct a comparative analysis on the amino acid sequences of human thymosin α1 and chicken thymosin α propeptide, and select the corresponding amino acid sequences of both as the chicken thymosin α1 sequence for protein expression. The amino acid sequence of chicken thymosin α1 is shown as SEQ ID NO.1.

[0021] (2) Construction of expression plasmid: Optimize the DNA sequence of chicken thymosin α1 according to the codon preference of Escherichia coli, commission a gene synthesis company to synthesize SEQ ID NO.2, and clone it between the BamHI and NotI sites of the pET28a plasmid, named pET28a-Tα1.

[0022] (3) Construction of recombinant expression strain: Transform the pET28a-Tα1 plasmid into Escherichia coli BL21 competent cells, pick a single colony for PCR identification of positive transformants, named E.coli BL21 / pET28a-Tα1.

[0023] (4) Expression and identification of target protein: Pick a single colony of E.coli BL21 / pET28a-Tα1, inoculate it into a 10 ml LB culture medium containing kanamycin, and culture it with shaking at 37 °C until the OD600nm reaches 0.6 - 0.8. Add 0.5 mol / L α-lactose to a final concentration of 0.03 mol / L, and culture it at 37 °C for another 6 h. After taking it out, centrifuge at 12000 r / min for 10 min to collect the bacteria. Add 10 times the volume of PBS based on the weight of the bacteria for bacterial cell suspension, and disrupt the cells by ultrasonic wave. Centrifuge at 12000 r / min for 15 min at 4 °C, and treat the supernatant and precipitate with SDS-PAGE loading buffer respectively, and perform SDS-PAGE and Western blot analysis.

[0024] (5) Purification of recombinant protein: Add 10 times the volume of PBS based on the weight of the bacteria for bacterial cell suspension, stir with a magnetic stirrer for 30 min until the precipitate is completely dissolved, then fully lyse the bacteria by ultrasound (performed on ice), centrifuge at 12000 r / min for 10 min, transfer the supernatant to a new tube, filter it through a 0.22 μm filter, and add it to a Ni-NTA chromatography column treated with Binding buffer, shake it gently at room temperature for 30 min, then let it stand at room temperature until the resin completely settles, and let the liquid flow out naturally. Subsequently, wash the chromatography column successively with 10 column volumes of Binding buffer and 6 column volumes of Washing buffer (8 M urea, 0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9), and finally elute the target protein with 6 column volumes of Elution buffer (6 M urea, 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), collect the eluate, take an appropriate amount of the eluate for SDS-PAGE electrophoresis detection, and store the purified protein at -20 °C for standby.

[0025] Example 2: Expression of chicken thymosin α1 mutant protein

[0026] (1) Design of the recombinant chicken thymosin α1 mutant sequence The charge carried by a protein is an important factor affecting renal clearance. Since the basement membrane of the kidney is negatively charged, under the same conditions, cationic polypeptides are often cleared faster. To extend the half-life of recombinant chicken thymosin α1 in chickens, the amino acid sequence of chicken thymosin α1 was analyzed, and the positively charged amino acid K at position 21 was mutated to the negatively charged amino acid D. The amino acid sequence of the chicken thymosin α1 mutant is shown in SEQ ID NO.3.

[0027] (2) Construction of the expression plasmid The DNA sequence of the chicken thymosin α1 mutant was optimized according to the codon preference of Escherichia coli, and SEQ ID NO.4 was synthesized by a gene synthesis company and cloned between the BamHI and NotI sites of the pET28a plasmid, named pET28a-Tα1M.

[0028] (3) Construction of the recombinant expression strain The pET28a-Tα1M plasmid was transformed into Escherichia coli BL21 competent cells, and single colonies were picked for PCR identification of positive transformants, named E.coli BL21 / pET28a-Tα1M.

[0029] (4) Expression and identification of the target protein Single colonies of E.coli BL21 / pET28a-Tα1M were picked and inoculated into 10 ml of LB medium supplemented with kanamycin, and cultured with shaking at 37 °C until the OD600nm reached 0.6 - 0.8. 0.5 mol / L α-lactose was added to a final concentration of 0.03 mol / L, and the culture was continued at 37 °C for 6 h. After that, it was centrifuged at 12000 r / min for 10 min to collect the bacteria. The bacteria were suspended in PBS at 10 times the volume of the bacterial weight and disrupted by sonication. Centrifugation was carried out at 4 °C and 12000 r / min for 15 min, and the supernatant and precipitate were treated with SDS-PAGE loading buffer, and SDS-PAGE and Western blot analyses were performed.

[0030] (5)Purification of the recombinant protein: Add PBS with a volume 10 times the weight of the bacterial cells to suspend the cells. Stir with a magnetic stirrer for 30 min until the precipitate is completely dissolved. Then, lyse the bacterial cells thoroughly by sonication (performed on ice). Centrifuge at 12,000 r / min for 10 min. Transfer the supernatant to a new tube, filter it through a 0.22-μm filter, and add it to a Ni-NTA chromatography column treated with Binding buffer. Oscillate at low speed at room temperature for 30 min, and then let it stand at room temperature until the resin completely settles. Let the liquid flow out naturally. Subsequently, wash the chromatography column thoroughly with 10 column volumes of Binding buffer and 6 column volumes of Washing buffer (8 M urea, 0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9) in sequence. Finally, elute the target protein with 6 column volumes of Elution buffer (6 M urea, 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Collect the eluate, take an appropriate amount of the eluate for SDS-PAGE electrophoresis detection, and store the purified protein at -20°C for later use.

[0031] Example 3: Expression of the tandem double-copy protein of the chicken thymosin α1 mutant

[0032] (1)Design of the tandem double-copy protein sequence of the chicken thymosin α1 mutant: To overcome the drawback that chicken thymosin α1 is easily cleared by the kidneys due to its molecular weight being less than the kidney filtration cut-off size, a tandem double-copy sequence of the chicken thymosin α1 mutant was designed, with an SG flexible peptide connecting the two copies. The amino acid sequence is shown in SEQ ID NO.5.

[0033] (2)Construction of the expression plasmid: Entrust a gene synthesis company to synthesize SEQ ID NO.6 and clone it between the BamHI and NotI sites of the pET28a plasmid, named pET28a-Tα1M2.

[0034] (3)Construction of the recombinant expression strain: Transform the pET28a-Tα1M2 plasmid into Escherichia coli BL21 competent cells. Pick a single colony for PCR identification of positive transformants, named E.coli BL21 / pET28a-Tα1M2.

[0035] (4)Expression and identification of the target protein Pick a single colony of E. coli BL21 / pET28a-Tα1M2 and inoculate it into 10 ml of LB culture medium supplemented with kanamycin. Incubate with shaking at 37°C until the OD600nm reaches 0.6 - 0.8. Add α-lactose with a final concentration of 0.03 mol / L to a final concentration of 0.5 mol / L, and then incubate at 37°C for another 6 h. After taking out, centrifuge at 12,000 r / min for 10 min to collect the bacteria. Add PBS with a volume 10 times the weight of the bacteria to suspend the bacteria, and then break the bacteria by ultrasonic wave. Centrifuge at 12,000 r / min at 4°C for 15 min. Treat the supernatant and precipitate with SDS-PAGE loading buffer respectively, and perform SDS-PAGE and Western blot analysis.

[0036] (5)Purification of the recombinant protein Add PBS with a volume 10 times the weight of the bacteria to suspend the bacteria. Stir with a magnetic stirrer for 30 min until the precipitate is completely dissolved, then fully lyse the bacteria by ultrasound (performed on ice). Centrifuge at 12,000 r / min for 10 min, transfer the supernatant to a new tube, filter it through a 0.22 μm filter, and then add it to a Ni-NTA chromatography column treated with Binding buffer. Oscillate at low speed at room temperature for 30 min, and then let it stand at room temperature until the resin completely settles, and let the liquid flow out naturally. Subsequently, wash the chromatography column successively with 10 column volumes of Binding buffer and 6 column volumes of Washing buffer (8 M urea, 0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9). Finally, elute the target protein with 6 column volumes of Elution buffer (6 M urea, 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Collect the eluate, take an appropriate amount of the eluate for SDS-PAGE electrophoresis detection, and store the purified protein at -20°C for standby.

[0037] Example 4: Determination of the activity of recombinant thymosin by E-rosette test

[0038] Take the thymus of a chicken, prepare a lymphocyte suspension, and remove the E receptor by water bath at 45°C for 30 min (dilute and count, final concentration is 3×10 6 ~5×10 6(cells / mL). They were placed in EP tubes, 0.2 mL per tube. 0.1 mL of thymosin α1 from chicken breast, thymosin α1 mutant from chicken breast, and double-copy thymosin α1 mutant from chicken breast samples diluted to 0.2 mg / mL were added respectively. For the control, 0.1 mL of Hank's solution was added. They were incubated at 37 °C for 1 hour, then 0.2 mL of sheep red blood cell suspension (counted, final concentration 10 times that of the E receptor-depleted T lymphocyte suspension) was added, shaken well, centrifuged at 500 r / min for 3 min, and placed in a 4 °C refrigerator overnight. The next day, they were taken out, the supernatant was discarded, 1 drop of fixing solution was added to each tube, shaken gently, left standing for 10 min, 2 drops of Giemsa staining solution were added and shaken well, and counting began after standing for 15 min. The larger, light blue cells in the microscope field of view were lymphocytes. The number of all lymphocytes on 16 large squares was counted (not less than 200), and the number of cells forming E rosettes was counted (lymphocytes binding 3 or more sheep red blood cells were counted as 1 rosette). The rosette percentage was obtained and the average value was taken. Sample viability = E rosette percentage of the test product determination tube - E rosette percentage of the control tube. A sample viability greater than 10% was considered qualified. Each group of this experiment was designed with 3 replicates.

[0039] It can be seen from Figure 2 that the E rosette percentage of the blank control was 11.33%, the E rosette percentage of thymosin α1 from chicken breast was 49.60%, the E rosette percentage of thymosin α1 mutant from chicken breast was 49.37%, and the E rosette percentage of double-copy thymosin α1 mutant from chicken breast was 48.93%. After calculation, the viability of thymosin α1 from chicken breast was 38.27%, the viability of thymosin α1 mutant from chicken breast was 38.04%, and the viability of [the relevant one] was 37.60%.

[0040] It shows that the expressed recombinant proteins of thymosin α1 from chicken breast, thymosin α1 mutant from chicken breast, and double-copy thymosin α1 mutant from chicken breast all have good biological activities. And the mutation of the positively charged amino acid K at the 21st position of the amino acid sequence of thymosin α1 from chicken breast to the negatively charged amino acid D did not affect its viability, and the activity of the tandem double-copy thymosin α1 mutant recombinant protein did not significantly decrease.

[0041] Example 5: Detection of the immune-enhancing activity of recombinant thymosin from chicken on chickens

[0042] (1) 40 15-day-old SPF chickens were randomly divided into 4 groups, 10 chickens in each group. Group A: Intramuscular injection of 0.4 mg of thymosin α1 protein in the chest muscle; Group B: Intramuscular injection of 0.4 mg of thymosin α1 mutant in the chest muscle; Group C: Intramuscular injection of 0.4 mg of double-copy thymosin α1 mutant in the chest muscle; Group D was the blank control without any treatment.

[0043] (2)At 1, 3, 5, and 7 weeks after thymopeptide injection, anticoagulated blood was collected from the wing vein. There were 10 samples in each group. Lymphocytes were separated using lymphocyte separation medium and washed twice with Hank's solution, and then resuspended in an appropriate amount of cell culture medium. After cell counting, the cell concentration was adjusted to 5×10 6 / mL. Lymphocytes were seeded into 96-well cell culture plates at 90 μL / well, and 10 μL of ConA with a concentration of 10 μg / mL was added. An unstimulated control was set for each sample, with 4 parallel wells, and a medium control was also set. After culturing at 5% CO2 and 37 °C for 48 h, 10 μL of MTS staining solution was added to each well, and then incubated at 5% CO2 and 37 °C for 2 h. The absorbance (OD492) at a wavelength of 492 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The average value of OD492 of the 4 parallel wells was taken. According to the formula: stimulation index SI = (sample OD492 - medium control OD492) / (unstimulated OD492 - medium control OD492), the stimulation index was calculated, which was used as an index to evaluate the ability of chicken thymopeptide to stimulate the proliferation of chicken peripheral blood lymphocytes.

[0044] It can be seen from Figure 3 that during the entire experimental period, the lymphocyte stimulation index SI of chickens in the blank control group was close to 1. At 1 week after thymopeptide injection, the lymphocyte stimulation index SI of chickens in the chicken thymopeptide α1, chicken thymopeptide α1 mutant, and double-copy chicken thymopeptide α1 mutant groups was about 1.85, and there was no significant difference among the three groups. At 3 weeks after thymopeptide injection, the SI value of the chicken thymopeptide α1 group decreased significantly to 1.422, the SI value of the chicken thymopeptide α1 mutant group decreased slightly to 1.72, and the SI value of the double-copy chicken thymopeptide α1 mutant group decreased slightly to 1.76. At 5 weeks after thymopeptide injection, the SI value of the chicken thymopeptide α1 group decreased significantly to 1.23, the SI value of the chicken thymopeptide α1 mutant group had decreased to 1.39, and the SI value of the double-copy chicken thymopeptide α1 mutant group remained at 1.61. At 7 weeks after thymopeptide injection, the SI value of the chicken thymopeptide α1 group had decreased to 1.08, approaching the control group level, the SI value of the chicken thymopeptide α1 mutant group decreased to 1.23, while the SI value of the double-copy chicken thymopeptide α1 mutant group still remained at a relatively high level of 1.43. It can be seen that the double-copy chicken thymopeptide α1 mutant has a more persistent effect on regulating the immune function of chickens than chicken thymopeptide α1 expressed by the natural sequence, and it is a long-acting thymopeptide that can be used as an immune enhancer.

Claims

1. A use of recombinant chicken thymosin in the preparation of a chicken immunopotentiator, characterized in that: The amino acid sequence of the recombinant chicken thymus peptide is SEQ ID NO.

5.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the recombinant chicken thymosin is SEQ ID NO.

6.

3. A chicken thymosin α1 mutant, characterized in that: The amino acid sequence of the chicken thymosin α1 mutant is SEQ ID NO.

3.

4. The chicken thymosin α1 mutant according to claim 3, characterized in that: The nucleotide sequence of the chicken thymosin α1 mutant is SEQ ID NO.4.

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