CN115850514A - Recombinant chicken interferon alpha protein, preparation method and application thereof - Google Patents
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Abstract
The invention discloses a recombinant chicken interferon alpha protein, a preparation method and application thereof, and belongs to the technical field of protein fusion. The recombinant chicken interferon alpha protein provided by the invention is formed by fusing chicken interferon alpha and albumin, and the amino acid sequence of the recombinant chicken interferon alpha protein is shown as SEQ ID NO. 2. The recombinant chicken interferon alpha protein prepared by the invention prolongs the half-life period of natural chicken interferon alpha, reduces the medication frequency, solves the problems of short half-life period, more medication frequency, high application cost and the like of the traditional interferon, can save the application cost, and can also reduce the manual operation frequency. In addition, the expression of the recombinant protein can be realized through temperature induction, the use of an inducer is avoided, and the production cost is also saved.
Description
Technical Field
The invention belongs to the technical field of protein fusion, and particularly relates to recombinant chicken interferon alpha protein, a preparation method and application thereof.
Background
The disease which is the most harmful to the chicken industry at present is infectious disease, especially virus disease in the infectious disease, and causes serious economic loss to the breeding industry. The ministry of agriculture forbids the use of antiviral chemical drugs such as ribavirin and amantadine in the breeding industry as early as 2005, so that western medicines are greatly limited in application. Viral infectious diseases are mainly prevented by vaccines at present, however, pathogeny on a breeding site is complex, complete protection of chicken flocks is difficult to form, infectious virus diseases such as newcastle disease, avian influenza, infectious bronchitis of chickens, marek's disease and the like sometimes occur, and serious threat is brought to the breeding industry.
Interferons (IFNs) are cytokines that induce animal cells to produce a wide variety of antiviral proteins, and have the functions of inhibiting virus reproduction, regulating body immunity, and resisting tumors. IFN genes are classified into type I and type II, and type I IFN is classified into alpha and beta, etc. Interferon alpha (IFN- α) is a cytokine produced by immune cells of the body, and is a group of structurally similar, functionally similar, low-molecular glycoproteins produced by immune cells through an anti-viral response when the body is infected with a virus. Interferons play a very important role in the immune system of the body. In recent years, many reports of interferon preparation by adopting a genetic engineering technology exist, but only canine interferon is currently approved for really realizing industrialization, and the main reason is that the problems of high application cost of cultured animals and the like are key factors influencing the industrialization. Meanwhile, no matter natural interferon or recombinant interferon generally has the problems of short half-life, more administration times, increased application cost, increased labor cost and the like.
Disclosure of Invention
The invention aims to realize the expression of recombinant fusion protein by constructing the fusion gene of chicken interferon alpha and albumin, cloning the fusion gene to an expression vector and inducing the temperature, thereby prolonging the half-life period of the natural chicken interferon alpha, reducing the frequency of medication and saving the production cost of an inducer used for expression.
The technical scheme of the invention is as follows:
the invention provides a recombinant chicken interferon alpha protein, which is formed by fusing chicken interferon alpha and albumin, and the amino acid sequence of the recombinant chicken interferon alpha protein is shown as SEQ ID NO. 2.
The expression gene of the recombinant chicken interferon alpha protein is shown as SEQ ID NO. 1.
The invention provides a recombinant expression vector containing the recombinant chicken interferon alpha protein expression gene. Preferably, the recombinant expression vector is a pBV220 vector containing recombinant chicken interferon alpha protein expression genes.
The invention also provides a recombinant bacterium containing the recombinant chicken interferon alpha protein expression gene. Preferably, the recombinant bacterium is escherichia coli containing a recombinant chicken interferon alpha protein expression gene.
The invention provides a preparation method of the recombinant chicken interferon alpha protein, which comprises the following steps: constructing the recombinant chicken interferon alpha protein expression gene into an expression vector to form a recombinant expression vector; transforming the recombinant expression vector into escherichia coli to obtain recombinant bacteria containing recombinant chicken interferon alpha protein expression genes; inducing the expression of the recombinant bacteria by using temperature, and purifying to obtain the recombinant chicken interferon alpha protein.
In the preparation method, the induction temperature is selected from 41-42 ℃. Preferably, the induction temperature is 42 ℃.
The specific method for inducing the expression of the recombinant bacteria by using the temperature comprises the following steps: culturing the recombinant bacteria in LB culture medium containing ampicillin, wherein the culture temperature is 30-32 ℃, when the OD value is 0.6, the culture temperature is increased to 41-42 ℃, and the recombinant bacteria are induced for 4-6 h to express.
The invention provides application of the recombinant chicken interferon alpha protein in preparation of a preparation with broad-spectrum antiviral effect.
The invention provides an interferon preparation for chicken, which contains the recombinant chicken interferon alpha protein; the formulation also preferably comprises other pharmaceutically acceptable adjuvants, such as potency stabilising protectants and the like.
The beneficial effects of the invention are as follows:
the recombinant chicken interferon alpha protein prepared by the invention prolongs the half-life period of natural chicken interferon alpha, reduces the medication frequency, solves the problems of short half-life period, more medication frequency, high application cost and the like of the traditional interferon, can save the application cost, and can also reduce the manual operation frequency. In addition, the expression of the recombinant protein can be realized through temperature induction, the use of an inducer is avoided, and the production cost is also saved.
Drawings
FIG. 1 is a double restriction enzyme identification electrophoresis diagram of recombinant plasmid; wherein, lane M: DNA Marker DL10000; lane 1: the recombinant plasmid double enzyme digestion electrophoresis band, wherein the upper band is a vector fragment with the size of 3600bp; the lower band is a fusion gene fragment with the size of 2349bp;
FIG. 2 shows the result of SDS-PAGE electrophoresis; wherein, lane M: a protein Marker; lane 1: empty bacterial cells not induced at 42 ℃; lane 2: inducing at 42 deg.c to obtain empty bacteria; lane 3: the recombinant bacteria were not subjected to 42 ℃ induction, and the bacterial cells were disrupted to obtain whole cells, lane 4: after the recombinant bacteria are induced for 5 hours at 42 ℃, the thalli are crushed to obtain whole bacteria;
FIG. 3 shows the result of Western Blot identification of recombinant proteins; wherein, lane 1: empty bacteria control; lane 2: inducing and breaking the recombinant bacteria to obtain whole bacteria; lane 3: and (4) precipitating the recombinant strain after induced crushing.
Detailed Description
In the invention, the design and synthesis of target genes, the construction of recombinant expression vectors and the construction of recombinant bacteria can be realized by conventional technical means. Other terms used in the present invention have meanings commonly understood by those of ordinary skill in the art unless otherwise specified. The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention, but not to limit the scope of the invention in any way.
Example 1
Preparation of recombinant protein:
(1) Design and Synthesis of Gene of interest
Designing a recombinant protein gene according to the gene sequence of the chicken interferon alpha gene-connecting peptide-albumin, and adding BamHI and SalI enzyme cutting sites at two ends of the gene respectively to form a target gene. The designed target gene is then handed over to the company of Biotechnology engineering (Shanghai) GmbH for direct synthesis. The sequence of the gene of interest is shown below:
SEQ ID NO:1：
the amino acid sequence of the expressed protein of the target gene is shown as follows:
SEQ ID NO:2：
(2) Construction of recombinant expression vector and recombinant bacterium
The pBV220 vector is subjected to double enzyme digestion by BamHI/SalI, is recovered, is connected with a target gene, is transformed into DH5 alpha escherichia coli competent cells, is coated on an LB culture medium plate containing 100 mu g/mL ampicillin for overnight culture, a single colony growing on the LB plate is taken to identify the target gene through PCR, and a positive clone bacterium plasmid is subjected to double enzyme digestion identification, and is identified as positive, which indicates that the construction of the expression vector is successful. As shown in FIG. 1, the recombinant expression vector was successfully constructed.
(3) Temperature induced recombinant bacteria expression and identification
Selecting the recombinant colony identified as the positive clone, streaking the recombinant colony on an LB culture medium plate containing ampicillin for overnight, selecting a single colony, inoculating the single colony in an LB culture medium containing 100 mu g/mL ampicillin for culture, increasing the culture temperature from 32 ℃ to 42 ℃ when the OD value is measured to be about 0.6, continuing induction culture for 5h, and collecting bacteria.
On the basis of the above expression method, the following controls were set: i, no induction at 42 ℃ is carried out on the empty bacterium thallus; II, inducing the air bacterium thallus at 42 ℃; III, the recombinant bacteria are not induced at 42 ℃.
Through SDS-PAGE electrophoresis detection, as shown in figure 2, compared with the empty bacterium, the recombinant bacterium has a concentrated new protein band at about 87KD, which shows that the recombinant bacterium can be promoted to successfully express the recombinant interferon alpha protein through temperature induction.
Western Blot was performed on the recombinant bacteria induced at 42 ℃ using a murine anti-chicken interferon- α (1 diluted 3500) as the primary antibody and a goat anti-mouse IgG-HRP (1 diluted 5000) as the secondary antibody. The test results are shown in fig. 3: the recombinant chicken interferon alpha sample can perform specific reaction with the chicken interferon alpha resisting monoclonal antibody, and a specific band appears at about 87 KD.
4. Preparation of crude recombinant protein product
And (3) crushing and homogenizing the collected bacteria in a high-speed tissue homogenizer, repeating the steps for 2 to 3 times, centrifuging the bacteria at 12000r/min for 10min, removing supernatant, and collecting precipitates to obtain crude recombinant protein.
5. Purification of recombinant proteins
Protein purification was performed using a GEAKTA pure purifier. Packing the column by nickel ion chelating affinity chromatography, and cleaning Ni with clear water 2+ Chelating affinity chromatography column, and Binding Buffer (20 mmol/L Na) 2 HPO 4 ·12H 2 O,500mmol/L NaCl,20mmol/L imidazole, pH = 8.0), detecting the conductivity value and the 280nm wavelength absorption value on line, starting to load the sample after the two are stable, loading the crude recombinant protein on a chromatographic column by using a loading pump, then loading the crude recombinant protein on the chromatographic column by using a Binding Buffer, and washing off the hybrid protein which is not combined with the chromatographic column until A280 is stable. Then use Elution Buffer (20 mmol/L Na) 2 HPO 4 ·12H 2 O,500mmol/L NaCl,500mmol/L imidazole, pH = 8.0) collected eluted eggsWhite, namely the purified recombinant chicken interferon alpha protein.
The protein content can be detected by Lowry method, and the protein concentration is not less than 20 mug/mL, which is qualified. The purified recombinant protein can be filtered and sterilized by a 0.22 mu m filter, sterilized and then can be aseptically subpackaged and stored at 2-8 ℃.
(ii) detection of biological Activity
The titer units were calculated as 50% lesion using a VSV/Wish system using a cytopathic inhibition method. Culturing Wish cells in 96-well microcyte culture plates with DMEM nutrient solution at 5% CO 2 Culturing at 37 deg.C for 12-24 hr in incubator, growing into good monolayer, adding recombinant chicken interferon alpha protein solution with different dosage (i.e. dilution in Table 1), and removing CO at 37 deg.C and 5% 2 Culturing for 24h under the condition. A normal cell control hole and a virus control hole are arranged in each test and are used as a normal cell control and a cell control with pathological changes. The mixture is discarded after 24h and then inoculated with 100TCID 50 VSV virus. At 37 ℃ C, 5% CO 2 Culturing under the condition, and judging the result when more than 75% of cells of the virus control hole have lesions (+++).
Inhibition of VSV cytopathic inhibition by fusion proteins the cytopathic inhibition was examined as shown in Table 1. Wherein, in Table 1, the interferon is diluted at 4 -5 For example, it refers to a 4-fold dilution at the time of loading interferon, 4 -5 Compared with the original preparation concentration (20 mug/mL), the concentration is equivalent to 4 -5 And (4) performing double dilution. The number of wells for inhibiting lesion accumulation represents the sum of the number of wells for inhibiting cytopathic effect accumulated from the high dilution listed on the table to the dilution under each sparsity; the number of lesion wells totaled represents the low dilution listed on the table for each dilution to the total number of wells in which cytopathic effect accumulated at that dilution.
TABLE 1
The titer is determined to be more than or equal to 1.39 multiplied by 10 according to the table 1 4 IU/mL. The calculation method is as follows:
definition of interferon potency: the potency was calculated by the Reed-Muench method, with interferon units expressed as log4 and log4 converted to the reciprocal of log 10. The reciprocal of the highest dilution of interferon capable of protecting half of cells from being attacked and destroyed by virus is the interferon titer.
Specifically, the method comprises the following steps: the sum of the log of the interferon dilution (log 4X) + distance ratio (assumed as Y value) that inhibits 50% of cytopathic effects is the dilution of interferon to 4 -Y Half of the cells are protected from damage. The dilution X is the titer of the sample to be detected. The formula for the distance ratio is as follows:
through the calculation, the method has the advantages that,
therefore, the logarithm of the dilution of interferon (log 4X) capable of inhibiting 50% of cytopathic effect is 6+0.88, i.e., the dilution of interferon to 4 -6.88 Half of the cells are protected from damage. The dilution X is the titer of the sample to be tested. Namely: log4X =6.88, using the logarithmic base-conversion formula: lgX =6.88log4, X =13873IU/mL is obtained from a log and inverse log table.
(II) half-life measurement
The relation between the blood concentration and the time of the interferon alpha is measured by an HPLC method, and pharmacokinetic parameters of the recombinant interferon alpha protein in chicken bodies are calculated by using Origin software for processing.
20 laying hens (half of the male and the female) with approximately the same weight of 21 days old are taken, 1mL (20 mu g/mL) of recombinant interferon alpha protein is injected subcutaneously into the neck, blood is collected under the wings of 1h, 4h, 8h, 16h, 24h, 48h, 96h, 144h, 192h, 240h and 320h respectively, the laying hens are placed in a 37 ℃ incubator and kept still for 30min, and the concentration of the interferon alpha is measured by HPLC. The concentrations of recombinant interferon alpha protein in the serum of the laying hens at different time points are shown in the following table 2:
TABLE 2
| Time (h) | Concentration (ng/ml) |
| 0 | 0 |
| 1 | 5.93±0.09 |
| 4 | 26.21±0.05 |
| 8 | 61.99±0.07 |
| 16 | 79.23±0.06 |
| 24 | 86.77±0.12 |
| 48 | 74.22±0.08 |
| 96 | 59.27±0.11 |
| 144 | 42.31±0.09 |
| 192 | 33.54±0.07 |
| 240 | 22.67±0.13 |
| 320 | 18.87±0.04 |
Through calculation of Origin software, the kinetic parameters in the serum of the laying hens are as follows: the absorption half-life T (1/2 alpha) is 8.616 +/-2.100, and the elimination half-life T (1/2 beta) is 158.32 +/-24.04. The results show that the recombinant chicken interferon alpha protein has longer half-life period which is up to 158h. Generally, the half-life period of exogenous interferon in vivo is only 2-6 h (see Wanglihong et al, "the rule of growth and contraction of interferon gamma in chicken body"), so that the half-life period of the recombinant chicken interferon alpha protein is at least improved by about 26 times compared with that of the common interferon.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention will still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. A recombinant chicken interferon alpha protein is characterized in that the recombinant chicken interferon alpha protein is formed by fusing chicken interferon alpha and albumin, and the amino acid sequence of the recombinant chicken interferon alpha protein is shown as SEQ ID NO. 2.
2. The expression gene of the recombinant chicken interferon alpha protein of claim 1 is shown as SEQ ID NO. 1.
3. A recombinant expression vector containing the recombinant chicken interferon alpha protein expression gene of claim 2.
4. A recombinant bacterium containing the recombinant chicken interferon alpha protein expression gene of claim 2.
5. The method for preparing the recombinant chicken interferon alpha protein of claim 1, which is characterized by comprising the following steps: constructing the recombinant chicken interferon alpha protein expression gene into an expression vector to form a recombinant expression vector; transforming the recombinant expression vector into escherichia coli to obtain recombinant bacteria containing recombinant chicken interferon alpha protein expression genes; inducing the expression of the recombinant bacteria by using temperature, and purifying to obtain the recombinant chicken interferon alpha protein.
6. The method according to claim 5, wherein the induction temperature is selected from 41 to 42 ℃.
7. The method of claim 6, wherein the induction temperature is selected from 42 ℃.
8. The method according to claim 5, wherein the specific method for inducing expression of the recombinant bacteria by using temperature is as follows: culturing the recombinant bacteria in LB culture medium containing ampicillin, wherein the culture temperature is 30-32 ℃, when the OD value is 0.6, raising the culture temperature to 41-42 ℃, and carrying out induced culture for 4-6 h, thereby inducing the expression of the recombinant bacteria.
9. Use of the recombinant chicken interferon alpha protein of claim 1 in the preparation of a preparation with broad-spectrum antiviral effect.
10. An interferon preparation for chickens, comprising the recombinant chicken interferon alpha protein according to claim 1.
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| CN103290021A (en) * | 2013-03-19 | 2013-09-11 | 安徽九川生物科技有限公司 | A preparation method for recombinant chicken interferon alpha |
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