CN103059123B - A kind of recombinant porcine interferon beta 1 and its coding gene and expression method - Google Patents

Abstract

The invention provides recombinant porcine interferon beta1, an encoding gene and an expression, purification and inclusion body refolding method and belongs to the field of biological genetic engineering. The recombinant porcine interferon beta1 has wide medical prospect in the field of veterinary medicine as a nonspecific broad-spectrum anti-viral biological preparation, but has the problems of shortage of throughput, high price, different drug specifications, and the like just as most genetic engineering drugs. In order to obtain a lot of recombinant porcine interferon beta1, an escherichia coli expression system is adopted to carry out heterologous expression on the recombinant porcine interferon beta1 gene optimized by a codon. In addition, an inclusion body purifying and refolding method of the recombinant porcine interferon beta1 is also provided by the invention aiming at the problem that the porcine interferon beta1 in a prokaryotic expression system is mostly expressed in a form of an inclusion body, so that the prepared porcine interferon beta1 has high activity and reaches the standard of industrial production.

Description

A kind of recombinant porcine interferon beta 1 and its coding gene and expression method

technical field

The invention belongs to the field of bioengineering genes, and relates to a recombinant porcine interferon beta 1 and its encoding gene, as well as its expression, purification and inclusion body renaturation methods.

Background technique

Interferon (Interferon, IFN) is a group of active proteins (mainly glycoproteins) with multiple functions, and is a cytokine produced by monocytes and lymphocytes. They have broad-spectrum anti-virus, affect cell growth, differentiation, regulation of immune function and other biological activities on the same kind of cells. According to the source of IFN, that is, animal species, cell type, the nature of the inducer and the induction conditions, it can be divided into three types: α, β, and γ. Among them, interferon β is mainly produced by fibroblasts, which can enhance the expression of HLAI and class II antigens on the cell surface in vivo, increase the levels of serum nephrin and β ₂ -microglobulin, enhance the viability of NK cells and ADCC, and enhance the function of peripheral lymphocytes. 2',5'-Oligadenosine synthase activity. Interferon β has been clinically applied in the treatment of multiple sclerosis, hepatitis B, hepatitis C, etc.

my country is a large pig-producing country, and various porcine viral infectious diseases such as porcine circovirus disease, porcine reproductive and respiratory syndrome, and porcine pseudorabies have brought huge economic losses to the pig industry. Although various swine disease vaccines are widely inoculated in my country at present, the disease epidemic cannot be effectively controlled. Interferon β has a strong antiviral effect and immunomodulatory activity. Like IFN-α, it has a broad-spectrum antiviral effect and can be used as a substitute for interferon α, and the degree of inhibition of interferon β on viruses will vary depending on the virus. For example, interferon beta has a better anti-SARS-CoV effect than interferon alpha; interferon beta is also significantly better than interferon gamma in acid, alkali, and heat tolerance. At the same time, IFN-β can also inhibit the proliferation of fibroblasts, epithelial cells, endothelial cells and hematopoietic cells, inhibit and kill tumor cells, and regulate the immune system, so it is more and more widely used in the field of veterinary medicine. However, there are obvious species differences in interferon β, and it is far from meeting the market requirements only by extracting it from live pigs. Genetically engineered interferon β for animals has no drug residues and no side effects, and is favored by clinical veterinarians and farmers. However, most similar drugs on the market still face problems such as insufficient production, uneven quality levels, and high prices. Therefore, this patent firstly provides a low-cost, stable-quality expression system and expression method capable of expressing a large amount of recombinant porcine interferon β1.

The prokaryotic expression system was the first to be used for research, and it is also the most mature expression system currently available. The main method of this technology is to transform bacteria (usually Escherichia coli) with the vector (usually a plasmid) cloned into the DNA fragment of the target gene, induce and finally purify the required target protein through IPTG. The advantage is that the gene expression product can be obtained in a short period of time, and the required cost is relatively low. This patent adopts Escherichia coli expression system to perform heterologous expression of recombinant porcine interferon β1, thereby obtaining a large amount of target protein. However, due to the lack of post-translational modification in the prokaryotic expression system, the expressed eukaryotic protein is often produced in the form of inclusion bodies with low activity. The renaturation of inclusion bodies is a very complicated process, which is not only closely related to the renaturation conditions and materials, but also largely depends on the properties of the protein itself. If the renaturation conditions are not suitable, it will lead to the mismatch of intramolecular disulfide bonds, and the formation of aggregates through covalent or hydrophobic bonding between molecules, so that the product will be precipitated, affecting the yield, and reducing the specific activity of the recombinant protein. rate, affecting product quality. Therefore, another technical problem to be solved in this patent is to adopt appropriate conditions to renature the inclusion body of the recombinant porcine interferon β1 produced by the E. coli expression system to obtain a product with higher specific activity.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a method for highly expressing recombinant porcine interferon beta 1 and its gene in Escherichia coli and methods for expression, purification and renaturation through codon optimization.

The invention provides a recombinant porcine interferon beta 1, the amino acid sequence of which is shown in SEQ ID NO:2.

The present invention provides the gene encoding the above-mentioned recombinant porcine interferon β1, the base sequence of which is shown in SEQ ID NO:1. The sequence is a codon-optimized sequence specially for the Escherichia coli expression system, which can significantly improve the expression efficiency of heterologous genes in host bacteria by comparison.

The present invention also provides a vector containing the above-mentioned gene encoding recombinant porcine interferon β1, the vector is preferably a prokaryotic expression plasmid, most preferably pET21b.

The present invention also provides an Escherichia coli strain containing the above-mentioned vector, preferably, the strain is selected from the Escherichia coli BL21 (DE3) strain.

The present invention also provides a method for expressing recombinant porcine interferon beta 1 in Escherichia coli, comprising the following steps:

The steps of this method are:

1. Pick an Escherichia coli colony containing the above-mentioned recombinant porcine interferon β1, insert LB culture medium, and cultivate overnight;

2. Take the overnight culture and inoculate it into LB medium, and culture it with shaking until the mid-logarithmic phase (A ₆₀₀ =1.0);

3. Add IPTG to the culture to 0.5-1.5mmol/L, induce expression at 37°C for 1-4h, and centrifuge to collect the E. coli bacterium pellet containing recombinant porcine interferon β1.

The LB culture solution all contains 50-100 μg/mL of ampicillin.

The present invention also provides a method for purifying inclusion bodies of recombinant porcine interferon beta 1, comprising the following steps:

1. Resuspend the collected Escherichia coli cells containing the induced recombinant porcine interferon β1, resuspend in pre-cooled PBS, and centrifuge at 4°C at high speed; repeat once.

2. Suck off the supernatant, weigh the precipitated bacteria, add lysis buffer BufferA3-10ml per gram (wet weight of bacteria), stir with a polished glass rod, and suspend the bacteria.

3. Add 3-10 μL of PMSF with a concentration of 100 mmol/L and 3-100 μL of lysozyme with a concentration of 100 mg/mL per gram (wet weight of bacteria), and stir on ice.

4. Break up the bacteria, place the sample on ice, sonicate, and centrifuge at 4°C at high speed, discard the supernatant.

5. Wash the pellet with Buffer B, and centrifuge at 4°C to precipitate inclusion bodies, repeat once.

6. The inclusion body precipitate was dissolved with denaturing buffer Buffer C, and stirred at room temperature for 30-60min.

7. After fully mixing, centrifuge at room temperature at high speed, discard the precipitate, and take the supernatant to obtain the denatured solution of recombinant porcine interferon β1.

The preferred steps of the purification method are as follows:

1. Resuspend the collected Escherichia coli pellet containing the induced recombinant porcine interferon β1 with pre-cooled PBS, and centrifuge at 12000 rpm/min for 15 min at 4°C; repeat once.

2. Suck off the supernatant, weigh the precipitated bacteria, add 5mL of lysis buffer BufferA per gram (wet weight of bacteria), stir with a polished glass rod, and suspend the bacteria.

3. Add 5 μL of PMSF with a concentration of 100 mmol/L and 5 μL of lysozyme with a concentration of 100 mg/mL to each gram (wet weight of bacteria), and stir on ice for 20 minutes.

4. Use a probe-type ultrasonic instrument to break up the bacteria, place the sample on ice, and ultrasonicate 120 times, with 5 seconds between each time and 5 seconds apart, and cycle three times. Wait for 2 minutes between each cycle and cool the sample, and wait for the sample to cool. Centrifuge at 12000 rpm/min for 15 min at 4°C and discard the supernatant.

5. The precipitate was washed with Buffer B, centrifuged at 12000rpm/min for 15min at 4°C to precipitate inclusion bodies, and repeated once.

6. The inclusion body precipitate was dissolved with denaturing buffer Buffer C, and stirred at room temperature for 30 minutes.

7. After fully mixing, centrifuge at 12,000 rpm/min for 15 min at room temperature, discard the precipitate, and take the supernatant to obtain the denatured solution of recombinant porcine interferon β1.

The present invention also provides the inclusion body renaturation method of the optimized recombinant porcine interferon beta 1, comprising the following steps:

Take an appropriate amount of the above-mentioned recombinant porcine interferon β1 denaturation solution dissolved in denaturation buffer Buffer C, measure its concentration, then dilute the protein concentration to 0.2 mg/mL with renaturation buffer Buffer D, and refold at 4°C to 24 After 1 hour, the recombinant protein solution after renaturation was passed through a 0.45 μm filter membrane to obtain a low-concentration recombinant porcine interferon β1 renaturation solution. Recombinant porcine interferon β1 powder is obtained by ultrafiltration, desalting, concentration, and low-temperature vacuum drying in a vacuum freeze dryer with a molecular weight cut-off of 10KDa.

The above-mentioned expression, purification, and renaturation methods of the present invention are the most effective methods for expressing recombinant porcine interferon beta 1 in the Escherichia coli expression system obtained through the inventor's repeated experiments and verifications, and the expression level of the method is high. , and the activity of the expression is higher after inclusion body renaturation. In particular, the gene sequence of the optimized recombinant porcine interferon beta 1 of the present invention is more suitable for the expression of Escherichia coli expression system, and the expressed recombinant porcine interferon beta 1 is much higher than the natural gene sequence of porcine interferon beta 1 expressed in Escherichia coli system expression.

The invention also provides the use of the recombinant porcine interferon beta 1 in the preparation of medicines for treating and preventing porcine reproductive and respiratory syndrome, swine flu and porcine blue-ear disease. During the treatment of piglet diseases, porcine interferon β1 can non-specifically exert a wide range of antiviral effects, improve the body's immune response and enhance the defense against viruses. At the same time, porcine interferon β1 can also be used in combination with other vaccines to reduce the adverse reactions of vaccines and enhance the overall anti-virus, bacteria, and parasite efficacy.

Description of drawings

Figure 1 shows the comparison of nucleotide sequences before and after codon optimization of recombinant porcine interferon β1

Among them, the even-numbered lines (that is, the lines corresponding to the "original sequence") are the nucleotide sequence of the natural gene of porcine interferon β1, that is, the sequence before codon optimization; the odd-numbered lines (that is, the lines corresponding to the "optimized sequence") are the nucleotide sequences of the present invention. The gene nucleotide sequence of recombinant porcine interferon beta 1, that is, the codon-optimized sequence.

Figure 2-a and Figure 2-b show the CAI index in the Escherichia coli expression host before and after codon optimization of recombinant porcine interferon β1.

Wherein, Fig. 2-a shows that the nucleotide sequence of the natural gene nucleotide sequence of porcine interferon beta 1 is calculated as 0.60 in the Escherichia coli expression host through a program; The CAI index in the Escherichia coli expression host was calculated to be 0.90 by the program.

Fig. 3-a and Fig. 3-b are the distribution area diagrams of the optimal codon frequency in the E. coli expression host before and after codon optimization of porcine interferon β1.

Wherein, Fig. 3-a shows the optimal codon frequency distribution region map of the nucleotide sequence of the natural gene of porcine interferon beta 1 in the Escherichia coli expression host, as can be seen from the figure: the nucleotide sequence of the natural gene of porcine interferon beta 1 The occurrence percentage of low-utilization codons is 15%; Fig. 3-b represents the optimal codon frequency distribution area diagram of the recombinant porcine interferon beta 1 codon of the present invention after optimization in the Escherichia coli expression host, and the optimized codon of the present invention The occurrence percentage of low utilization codons in the codon sequence of recombinant porcine interferon β1 was 0.

Fig. 4-a and Fig. 4-b are distribution area diagrams of average GC base content in E. coli expression hosts before and after codon optimization of recombinant porcine interferon β1.

Wherein, Fig. 4-a shows that the nucleotide sequence of the natural gene of porcine interferon beta 1 has an average GC base content of 46.45% in the E. coli expression host; Fig. 4-b shows the code of the optimized recombinant porcine interferon beta 1 of the present invention The average GC base content of the child in the E. coli expression host is: 47.27%.

Figure 5-a and Figure 5-b are the predicted secondary structures of mRNA before and after codon optimization of recombinant porcine interferon β1.

Fig. 5-a is the predicted secondary structure diagram of porcine interferon β1 natural gene mRNA, and Fig. 5-b is the predicted secondary structure diagram of recombinant porcine interferon β1 mRNA of the present invention after codon optimization.

Fig. 6 is a diagram showing the construction process of recombinant porcine interferon beta 1 expression plasmid.

Fig. 7 is an agarose gel electrophoresis picture of the recombinant porcine interferon β1 gene PCR product.

Among them, lane 1 is the pET21b vector digested with NdeI and XhoI; lane 2 is the 500bp DNA Ladder; lane 3 is the PCR product of recombinant porcine interferon β1 gene with NdeI and XhoI restriction sites at both ends.

Figure 8-a and Figure 8-b are the SDS-PAGE gel electrophoresis and the corresponding Western blot images of recombinant porcine interferon β1.

Figure 8-a is the SDS-PAGE gel electrophoresis image of recombinant porcine interferon β1.

Among them, lane 1 is (10-230kDa) wide range pre-stained protein loading marker; lane 2 is the lysate of Escherichia coli without adding IPTG-induced recombinant porcine interferon β1; lane 3 is adding IPTG-induced recombinant porcine interferon β1 E. coli lysate.

Figure 8-b is the western blot of recombinant porcine interferon β1.

Among them, lane 1 (10-230KDa) is a wide range of pre-stained protein loading markers, lane 2 is the recombinant porcine interferon β1 Escherichia coli lysate without adding IPTG; lane 3 is the recombinant porcine interferon β1 induced by adding IPTG E. coli lysate.

Fig. 9 SDS-PAGE gel electrophoresis diagram of optimized induction conditions for high expression of recombinant porcine interferon β1.

Among them, lane 1 is (10-230kDa) wide range pre-stained protein loading marker; lane 2 is 0.5mmol/L IPTG induced 1h E. coli lysate containing recombinant porcine interferon β1; lane 3 is 0.5mmol/L The lysate of E. coli containing recombinant porcine interferon β1 induced by IPTG for 2 hours; lane 4 is the lysate of E. coli induced by 0.5mmol/L IPTG for 3 hours; lane 5 is the lysate of E. coli induced by 0.5mmol/L IPTG for 4h Escherichia coli lysate containing porcine interferon β1; lane 6 is E. coli lysate containing recombinant porcine interferon β1 induced by 1mmol/LIPTG for 1h; lane 7 is Escherichia coli lysate containing recombinant porcine interferon β1 induced by 1mmol/L IPTG for 2h; Swimming lane 8 is the lysate of Escherichia coli containing recombinant porcine interferon β1 induced by 1mmol/L IPTG for 3h; Swimming lane 9 is the lysate of Escherichia coli containing recombinant porcine interferon β1 induced by 1mmol/L IPTG for 4h; 1h Escherichia coli lysate containing recombinant porcine interferon β1; lane 11 is 1.5mmol/L IPTG induced 2h containing recombinant porcine interferon β1 Escherichia coli lysate; lane 12 is 1.5mmol/L IPTG induced 3h containing recombinant porcine interferon lysate of Escherichia coli containing recombinant porcine interferon β1; lane 13 is the lysate of Escherichia coli induced by 1.5mmol/L IPTG for 4h.

Figure 10 is the SDS-PAGE electrophoresis of the recombinant porcine interferon beta 1 inclusion body after renaturation

Among them, lane 1 is a wide range of (10-230kDa) pre-stained protein loading marker; lane 2 is the whole bacterial lysate after induction; lane 3 is the inclusion body precipitation of recombinant porcine interferon β1 after the first washing of Buffer B ; Swimming lane 4 is the inclusion body precipitation of recombinant porcine interferon β1 after the second washing of Buffer B; Swimming lane 5 is the recombinant porcine interferon β1 after dilution and refolding

Detailed ways

The present invention will be further described below in conjunction with specific examples. It should be understood that the cited examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1 Recombinant porcine interferon beta 1 gene optimization design

1. Codon optimization

There are 64 genetic codes, but most organisms tend to use a subset of these. Those that are most frequently used are called optimal codons, and those that are not frequently used are called rare or low-usage codons. Virtually every organism commonly used for protein expression or production (including E. coli, yeast, mammalian cells, plant cells, and insect cells) exhibits some degree of difference or bias in codon usage. The expression efficiency of genes containing optimal codons in E. coli, yeast and Drosophila was significantly higher than that of genes containing low utilization codons. Therefore, in the heterologous expression system, the codon bias largely affects the expression of recombinant proteins. Gene synthesis using preferred codons and avoiding the use of rare codons is called codon optimization. The optimization process fully takes into account various complex factors that may be encountered at different stages of protein expression, such as: codon adaptation, mRNA structure, and different cis-elements during transcription and translation. Therefore, the gene design of porcine interferon β1 in the present invention includes not only codon optimization, but also mRNA structure correction, translation initiation site optimization and the like.

2. Codon preference optimization

Codon bias has been proven to be a very important factor in prokaryotic gene expression, which causes the utilization of the same codon between different organisms, protein expression levels and different parts of the same operon. Change. The main reason for this difference in preference is the difference in the amount of tRNAs available in different cells. Therefore, the best way to optimize the translation system is to maintain the balance between codon usage frequency and cognate tRNA. Expressing mammalian genes in E. coli is unpredictable and challenging. For example, there are few tRNA molecules corresponding to AGG and AGA in E. coli. This difference will obviously affect gene expression.

3. Replace low-utilization codons with common host codons

Generally, the lower the utilization rate of the codon contained in the gene in a specific host, the less the expression of the protein, even when the codon exists between the protein clusters or the N-terminus, the expression will be less. Under the premise of not changing the amino acid sequence, replacing low-utilization codons with common codons of the host can improve the expression level of functional proteins.

Expression inhibition occurs when codons from any source are less than 5% to 10% utilized in the host organism. When these low-utilization codons are adjacent or connected, the impact on protein expression is greater. Clusters of underutilized codons inhibit ribosome movement, an apparent mechanism by which genes cannot be expressed at appropriate levels. Ribosomes move faster when translating a nine-codon message with several or all low-use codons than when translating an equally long message without low-use codons slow. Even if the low-utilization codon cluster is at the 3' end, the message ends up being damaged by "crowding" by the ribosome, which goes back to the 5' end. The suppressive effect of a cluster of underutilized codons at the 3' end can be as large as that of an entire message consisting of underutilized codons. If the cluster of underutilized codons is located at the 5' end, the effect is an overall reduction in the number of starting ribosomes, leading to inefficiency of the messenger in protein synthesis. Removal of underutilized codons or codons that are easily misinterpreted as stop signals can prevent low or no expression.

4. Expression vector and transcription promoter

Although codon bias plays an important role in gene expression, the choice of expression vector and transcription promoter is equally important, and protein expression from N-terminal nucleotide sequences is critical for low-utilization codons and codons close to the start site. AUG is very sensitive. There is also a mutual influence between translation and mRNA stability, although reducing translation efficiency will make mRNA easier to be decomposed by endo-RNAses due to the lack of ribosome protection, but there is no complete explanation for the influence between them.

Other factors can also affect protein expression, including sequences that destabilize mRNA. Stabilization of mRNA secondary structure and molecules near the 5' end also has important effects on gene expression. Using the open reading frame upstream of the target gene in translation can successfully improve the expression efficiency of difficult genes.

According to the cDNA sequence of porcine interferon β1 (Sus scrofa interferon, beta1) published by GenBank (GenBank accession number: NM_001003923.1), the inventor optimized the codon of the gene to obtain the recombinant porcine interferon β1 gene of the present invention, As shown in SEQ ID No: 1.

The following is the codon optimization of recombinant porcine interferon β1. The parameters before and after optimization are compared as follows:

1. Codon Adaptation Index (CAI)

It can be seen from Figure 2-a that before codon optimization, the codon adaptation index (CAI) of the porcine interferon β1 natural gene in Escherichia coli was 0.60. It can be seen from Fig. 2-b that after codon optimization, the CAI index of the recombinant porcine interferon β1 gene of the present invention in Escherichia coli is 0.90. Usually, when CAI=1, it is considered that the gene is in the most ideal high-efficiency expression state in the expression system, and the lower the CAI index, the lower the expression level of the gene in the host, so it can be seen that the codon optimization is obtained The gene sequence can increase the expression level of the recombinant porcine interferon beta 1 gene in Escherichia coli.

2. Frequency of Optimal Codons (FOP)

It can be seen from Fig. 3-a that, based on the expression vector of E. coli, before codon optimization, the occurrence percentage of low-utilization codons in the natural gene sequence of porcine interferon β1 is 15%. This non-optimized gene contains rare codons in tandem that may reduce translation efficiency or even dismantle translation assemblies. It can be seen from FIG. 3-b that after codon optimization, the frequency of low-utilization codons in the recombinant porcine interferon β1 gene of the present invention in the E. coli system is 0.

3. GC base content (GC curve)

The ideal distribution area of GC content is 30%-70%, and any peak outside this area will affect transcription and translation efficiency to varying degrees. It can be seen from the comparison of the average GC base content distribution area map of the porcine interferon β1 gene in Figure 4-a and Figure 4-b that the average content of GC bases in the natural gene of porcine interferon β1 before optimization is shown in Figure 4-a It is 46.45%. Figure 4-b shows that the optimized sequence eliminates all bases outside the region with a GC content of 30%-70%, and finally the average GC base content of the optimized recombinant porcine interferon β1 is 47.26%. .

3. The conditions of cis-acting elements before and after optimization are as follows:

cis-acting element Optimized before optimization E. coli_RBS (AGGAGG) 0 0 PolyT(TTTTTT) 0 0 PolyA (AAAAAAA) 0 0 Ch site (GCTGGTGG) 0 0 T7Cis(ATCTGTT) 0 2

4. The situation of palindrome and repeated sequence before and after optimization is as follows:

5mRNA secondary structure prediction map

After DNA is transcribed into mRNA, since mRNA is a single-stranded linear molecule, the complementary base pairs meet through self-folding, forming a hairpin structure (Hairpin) through hydrogen bonding. The 5' hairpin can play a regulatory role in the initiation of translation.

However, if the hairpin structure is very long and the energy required for unzipping is high, it may affect translation. Therefore, the sequences that need to be expressed should try to avoid long and high-energy hairpin structures. After codon optimization, the predicted secondary structure of mRNA before and after codon optimization of porcine interferon β1 in Figure 5-a and Figure 5-b shows that the optimized 5' hairpin structure and the energy required for unzipping are more suitable for the purpose protein expression.

Embodiment 2: the expression plasmid construction of recombinant porcine interferon beta 1 gene

The fragment synthesized from the optimized recombinant porcine interferon β1 gene (as shown in SEQ ID No: 1) was constructed into the pUC57 plasmid (provided by Nanjing GenScript Technology Co., Ltd.) to obtain a long-term preservation plasmid. It is the pUC57-prIFNβ1 plasmid. Using the pUC57-prIFNβ1 plasmid as a template, the upstream and downstream primers were respectively introduced into NdeI and XhoI restriction sites for PCR amplification. The sequences of the primers used are as follows:

Upstream primers:

P1: CGGGAATTCCATATGATGTCCTATGATGTTCTGCG

Downstream primers:

P2: CCGCTCGAGTTAATTGCGCAGATAATCCGTC

The total volume of the reaction is 50 μL, in which 2.5 μL of each primer with a concentration of 10 μmol/L is added, and 1 μL of dNTP with a concentration of 10 mmol/L is added. The DNA polymerase used is Phusion High-Fidelity DNA polymerase (purchased from Theromo-Fisher scientific), 2 U/μL, Add 0.5 μL. The reaction conditions were 98°C for 5s, 55°C for 20s, and 72°C for 30s. After 25 cycles, the product was analyzed by 1.0% agarose gel electrophoresis, and the results showed that the size of the product was consistent with the expected size (498bp). (As shown in Figure 7)

The obtained gene product was purified with a DNA gel recovery kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.). After purification, it was digested with NdeI and XhoI (purchased from New England Biolabs), connected to pET21b plasmid (purchased from Merck) with T4 ligase (purchased from New England Biolabs), and transformed into DH5α competent cells (purchased from (from Beijing Tiangen Biochemical Technology Co., Ltd.), cultured overnight at 37°C on LB plates containing 100 μg/mL ampicillin (purchased from Amresco). On the second day, the positive clones were screened, sequenced and compared, and they were completely consistent with the expected sequence, and an expression plasmid of a form of recombinant porcine interferon β1 was obtained, which was denoted as pET21b-prIFNβ1.

Example 3 Expression and Identification of Recombinant Porcine Interferon β1 in Escherichia coli

Specific steps are as follows:

1. The pET21b-prIFNβ1 plasmid with correct sequencing alignment in Example 2 was transformed into a competent strain of Escherichia coli BL21 (DE3) (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and cultured overnight on an ampicillin plate at 37°C.

2. On the second day, pick 1-4 recombinant colonies containing the pET21b-prIFNβ1 plasmid, insert them into LB medium containing 100 μg/mL ampicillin, and culture them overnight at 37°C.

3. Take 50 μL of the overnight culture and insert it into 5 mL of LB induction medium containing 100 μg/mL ampicillin, and culture at 37°C with shaking.

4. The OD600 value of the bacterial solution was measured every 1 h after inoculation, and when the OD600=1.0, the expression was induced with 1 mmol/L IPTG (purchased from Amresco). At the same time, the Escherichia coli culture solution without adding IPTG was used as a negative control.

5. After 4 hours, collect the bacterial liquid, centrifuge at high speed (speed: 12000rpm/min) for 3min, wash the precipitate with pre-cooled PBS, add 5XSDS gel loading buffer, heat at 100°C for 10min, and centrifuge at room temperature at high speed (speed: 12000rpm/min ) for 1 min, take the supernatant. Escherichia coli culture fluid without adding IPTG is also processed according to this step.

6. Take 10 μL of culture samples obtained in step 5 without adding IPTG and adding IPTG induced culture samples, and analyze by 12% SDS-PAGE gel electrophoresis.

7. Electrophoresis at 8-15V/cm until bromophenol blue migrates to the bottom of the separation gel.

8. Coomassie Brilliant Blue staining and immunoblotting to observe the expression product bands, see Figure 8-a and Figure 8-b.

Embodiment 4 Recombinant porcine interferon beta 1 highly expressed induction condition optimization

Many studies have shown that the cell growth rate seriously affects the expression of foreign proteins, so the amount of inoculated bacteria, culture temperature, cell growth time before induction and cell density after induction must be controlled. Excessive growth or excessive growth will increase the interference of E. coli forming recombinant pigs Prime β1 inclusion body. Using three factors and four levels, an orthogonal table of IPTG concentration and induction time was established, through

SDS-PAGE gel electrophoresis analysis induced the expression of recombinant porcine interferon β1.

Specific steps are as follows:

1. The pET21b-prIFNβ1 plasmid with correct sequencing alignment in Example 2 was transformed into a BL21 (DE3) competent strain (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and cultured overnight on an ampicillin plate at 37°C.

2. The next day, pick the control bacteria and 1-4 recombinant colonies containing the pET21b-prIFNβ1 plasmid, insert them into the LB culture medium containing 100 μg/mL ampicillin, and culture them overnight at 37°C.

3. Take 50 μL of the overnight culture and insert it into 5 mL of LB induction medium containing 100 μg/mL ampicillin, and culture at 37°C with shaking.

4. After inoculation, the OD600 value of the bacterial solution was measured. When OD600=1.0, 0.5, 1.0, 1.5mmol/LIPTG concentration and time were added according to Table 1 to induce expression. At the same time, the Escherichia coli culture solution without adding IPTG was used as a negative control.

Table 1 expresses IPTG concentration and time conditions

5. After 1, 2, 3, and 4 hours, collect the recombinant porcine interferon β1 bacterial solution in turn, centrifuge at a high speed (rotational speed: 12000rpm/min) for 3 minutes, wash the precipitate with pre-cooled PBS, and obtain Escherichia coli containing induced recombinant porcine interferon β1 For precipitation, add 5XSDS gel loading buffer, heat at 100°C for 10min, centrifuge at room temperature for 1min at high speed (speed: 12000rpm/min), and take the supernatant. Escherichia coli culture fluid without adding IPTG is also processed according to this step.

6. Take 10 μL of the recombinant porcine interferon β1 samples treated in step 5 without adding IPTG and adding different concentrations of IPTG and different induction time conditions, and analyze by 12% SDS-PAGE gel electrophoresis.

7. Electrophoresis at 8-15V/cm until bromophenol blue migrates to the bottom of the separation gel.

8. Coomassie brilliant blue staining was used to observe the expression product band of recombinant porcine interferon β1. See Figure 9.

Gel imaging system TLC scanning analysis of expressed recombinant porcine interferon β1 content identification of recombinant porcine interferon β1 expression. Finally, it is determined that the induction condition suitable for the present invention is 1mmol/L IPTG, and the induction time is 4h.

Example 5 Purification and Refolding of Recombinant Porcine Interferon β1 Inclusion Body

1. Resuspend the Escherichia coli pellet containing the induced recombinant porcine interferon β1 obtained by washing the precipitate with pre-cooled PBS in Step 5 of Example 4, resuspend it with pre-cooled PBS, and centrifuge at 12000 rpm/min at 4°C for 15 min; repeat once.

2. Aspirate the supernatant, weigh the precipitated bacteria, add 5 mL of lysis buffer BufferA per gram (wet weight of bacteria), stir with a polished glass rod, and suspend the bacteria.

3. Add 5 μL of 100 mmol/L PMSF and 5 μL of 100 mg/mL lysozyme to each gram (wet weight of bacteria), and stir on ice for 20 minutes.

4. Use a probe-type ultrasonic instrument to break up the bacteria, place the sample on ice, and ultrasonicate 120 times, with an interval of 5 seconds for each time, and cycle three times. 4°C, 12000rpm/min, centrifuge for 15min.

5. Wash the pellet with Buffer B, centrifuge at 12000rpm/min at 4°C for 15min to precipitate inclusion bodies, and repeat once.

6. The inclusion body precipitate was dissolved with denaturing buffer Buffer C, and stirred at room temperature for 30 minutes.

7. After fully mixing, centrifuge at room temperature 12000rpm/min for 15min, discard the precipitate, and take the supernatant to obtain the denatured solution of recombinant porcine interferon β1.

8. Refold the denatured solution of recombinant porcine interferon β1 in step 7 by dilution refolding method.

Take an appropriate amount of recombinant porcine interferon β1 denaturation solution dissolved in denaturation buffer Buffer C, measure its concentration with Quick Start Bradford 1x Dye Reagent (Bio-rad, USA), and then dilute the protein concentration to 0.2mg/ mL, when refolding at 4°C for 24 hours, pass the refolded recombinant protein solution through a 0.45 μm filter membrane (MerckMillipore) to obtain a low-concentration recombinant porcine interferon β1 refolding solution. Ultrafiltration tubes with a molecular weight cut-off of 10KDa (Merck

Millipore Company) desalted, concentrated, and vacuum-dried at a low temperature in a vacuum freeze dryer (Beijing Sihuan Scientific Instrument Factory Co., Ltd.) to obtain recombinant porcine interferon β1 powder.

Each buffer was prepared according to the table below:

Table 2 buffer preparation

9. Use the washing buffer Buffer B in step 5 to wash the product twice and refold the product for SDS-PAGE electrophoresis analysis (as shown in Figure 10), and obvious bands can be seen in the target range.

Embodiment 6 Recombinant porcine interferon beta 1 biological activity assay

Porcine vesicular stomatitis virus (VSV, whose TCID ₅₀ is 5×107/100 μL, provided by the Veterinary Research Institute of Guangdong Academy of Agricultural Sciences) can infect pig kidney cells (PK-15, provided by the Veterinary Research Institute of Guangdong Academy of Agricultural Sciences), The invention uses the antiviral mechanism of porcine interferon β1 to detect the defense effect of porcine kidney cells on porcine vesicular stomatitis virus under the condition of porcine interferon β1, and obtains the antiviral effect of porcine interferon β1 by detecting the pathological changes of PK-15 cells. The protective effect curve of PK-15 cells, so as to measure the biological activity of recombinant porcine interferon β1.

1. Preliminary screening experiment of biological activity

Positive control substance solution preparation: take porcine interferon positive control substance (porcine genetically engineered recombinant cytokine: IFN-LLS-2, purchased from Hong Kong Manpu Animal Nutrition Co., Ltd. and measure its titer to 5×10 ⁴ U/mL ), after reconstitution according to the instruction manual, the MEM cell culture medium (Gibico product) containing 6% fetal bovine serum was used to dilute step by step by 10 times.

Preparation of recombinant porcine interferon β1 sample solution: Weigh 1 mg of recombinant porcine interferon β1 sample after renaturation, dissolve it in 500 μL cell culture medium, and then use cell culture medium to make 10-fold serial dilutions to obtain 2×10 ³ μg/mL, 2×10 ² μg/mL, 20 μg/mL, 2 μg/mL, 2×10 ^-1 μg/mL, 2×10 ^-2 μg/mL recombinant interferon β1 solution.

Add the above-mentioned different concentrations of recombinant porcine interferon β1 to each well of a 96-well plate, and then add 50 μL of freshly passaged PK-15 cell suspension (cell concentration is about 1.8×10 ⁶ to 2.2×10 ⁶ cells/mL), the total 100 μL of the system was incubated for about 24 hours at 37°C and 5% CO ₂ , and the PK-15 cells adhered and grew to a monolayer. The supernatant of the cell culture solution was discarded, and 100 μL of MEM culture solution containing 100 TCID50 of VSV virus and 2% fetal bovine serum was added to each well. At the same time, set up a negative control virus group (only add PK-15 cells and the same dose of virus, without adding recombinant porcine interferon β1) and a blank cell control group (only add PK-15 cells and cell culture medium, without adding recombinant porcine interferon β1 and virus). Cultivate for 24 hours at 37° C. and 5% CO ₂ , and observe the results when the cytopathy of the virus control wells is 90% to 100%. The amount of interferon that causes cytopathic effects in half of the wells is taken as a unit (denoted as "U").

The experimental results showed that the cells in the negative control virus group all had obvious lesions, and the cells in the blank cell control group grew normally. Compared with the positive control, when the concentration of recombinant porcine interferon β1 was 20 μg/mL～2×10 ^-1 μg/mL, the effect of VSV virus on the normal growth of PK-15 cells began to be eliminated.

2. Biological activity re-screening experiment

According to the preliminary screening results, adjust the experimental concentration of the positive control and recombinant porcine interferon β1:

Positive reference substance solution preparation: Take porcine interferon positive reference substance, reconstitute according to the instructions, dilute with cell culture medium to contain about 1000IU per 1ml, and dilute step by step according to 4 times.

Preparation of recombinant porcine interferon β1 sample solution: According to the preliminary detection results of the activity of the renatured sample in Example 5, the recombinant porcine interferon β1 obtained by renaturation was diluted to 20ug/mL with cell culture medium, and then diluted step by step by 4 times 20ug/mL, 5μg/mL, 1.25μg/mL, 3.13×10 ^-1 μg/mL, 7.81×10 ^-2 μg/mL, 1.95×10 ^-2 μg/mL, 4.88×10 ^-3 μg/mL , 1.22×10 ^-3 μg/mL solution of recombinant porcine interferon β1.

Add the above-mentioned different concentrations of recombinant porcine interferon β1 to each well of a 96-well plate, and then add 50 μL of freshly passaged PK-15 cell suspension (cell concentration is about 1.8×10 ⁶ to 2.2×10 ⁶ cells/mL), the total 100 μL of the system was incubated for about 24 hours at 37°C and 5% CO ₂ , and the PK-15 cells adhered and grew to a monolayer. The supernatant of the cell culture solution was discarded, and 100 μL of MEM culture solution containing 100 TCID50 of VSV virus and 2% fetal bovine serum was added to each well. At the same time, set up a negative control virus group (only add PK-15 cells and the same dose of virus, without adding recombinant porcine interferon β1) and a blank cell control group (only add PK-15 cells and cell culture medium, without adding recombinant porcine interferon β1 and virus). Cultivate for 24 hours at 37° C. and 5% CO ₂ , and observe the results when the cytopathy of the virus control wells is 90% to 100%. The amount of interferon that causes cytopathic effects in half of the wells is taken as a unit (denoted as "U").

The experimental results showed that the cells in the negative control virus group all had obvious lesions, and the cells in the blank cell control group grew normally. Take the amount of interferon that causes cytopathic changes in half of the wells as a unit (denoted as "U"), observe the number of cells that are not affected by the VSV virus in the presence of positive control under the condition of positive control, and determine the positive control interferon The titer is 3.2×10 ⁴ U/mL. In the same way, the number of PK-15 cells that were not affected by the VSV virus in the presence of recombinant porcine interferon β1 obtained after renaturation was observed under the microscope, and the titer of the renatured sample of recombinant porcine interferon β1 was calculated to be 4.8×10 ⁴ U/mL.

It can be seen that the activity of the recombinant porcine interferon beta 1 obtained after renaturation in the present invention is significantly higher than that of the interferon positive control sold on the market, and the preparation process is simple, and it has shown broad application in the prevention and treatment of piglet diseases. The application prospect makes it possible to realize the possibility of industrialized production of genetically engineered recombinant porcine interferon β1.

Claims (6)

1. an encoding gene for Recombinant Swine interferon beta 1, its base sequence is as shown in SEQ ID NO:1.

2. a carrier, described carrier has gene as claimed in claim 1.

3. carrier as claimed in claim 2, described carrier is pET21b.

4. intestinal bacteria, described intestinal bacteria have carrier claimed in claim 3.

5. intestinal bacteria as claimed in claim 4, described intestinal bacteria are BL21(DE3) bacterial strain.

6. an expression method for Recombinant Swine interferon beta 1, comprises the steps:

(1) the intestinal bacteria bacterium colony described in picking claim 4 or 5, access is containing antibiotic LB nutrient solution, overnight incubation;

(2) get overnight culture and transfer in containing in antibiotic fresh LB nutrient solution, shake and be cultured to mid-log phase A ₆₀₀=1.0;

(3) in culture, adding concentration is the IPTG of 0.5-1.5m mol/L, and 37 ℃, after abduction delivering 1-4 hour, centrifugal treating is collected the coli somatic precipitation that contains Recombinant Swine interferon beta 1.

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