CN114107316A

CN114107316A - Codon-optimized pig lambda 3 interferon coding gene and application thereof in preparation of pig lambda 3 interferon

Info

Publication number: CN114107316A
Application number: CN202111485819.8A
Authority: CN
Inventors: 商营利; 李艳洁; 崔灿
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-03-01

Abstract

The invention discloses a codon-optimized porcine lambda 3 interferon coding gene and application thereof in preparation of porcine lambda 3 interferon. In particular discloses a codon-optimized porcine lambda 3 interferon gene PoIFN lambda 3-MD and a method for preparing recombinant porcine lambda 3 interferon by using the gene. The expression capacity of the optimized gene PoIFN lambda 3-MD in host bacteria is far higher than that of the unoptimized gene PoIFN lambda 3-WT. The optimized PoIFN lambda 3-MD is inserted into a multiple cloning site of a vector pET30a (+) to obtain a recombinant plasmid, and the recombinant plasmid is introduced into escherichia coli to obtain a recombinant bacterium. The recombinant strain can be used for preparing a large amount of high-activity recombinant porcine lambda 3 interferon protein through induction culture. The gene, the recombinant plasmid and the recombinant bacterium provided by the invention have great economic value for the production of the recombinant porcine lambda 3 interferon, and also have great application value and wide application prospect for the live pig breeding industry.

Description

Codon-optimized pig lambda 3 interferon coding gene and application thereof in preparation of pig lambda 3 interferon

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a codon-optimized porcine lambda 3 interferon coding gene and application thereof in preparation of porcine lambda 3 interferon.

Background

In recent years, various diseases are caused frequently in high-density intensive live pig breeding modes in China, and particularly various viral diseases become key factors for restricting sustainable development of breeding industry in China. Various chemicals are used for treating the porcine viral diseases for a long time, but the drugs are easy to cause the drug residue to exceed the standard, and the search for new safe and residue-free antiviral drugs is imminent, so that the research and the development of novel drugs capable of effectively preventing and treating the livestock viral diseases have strong market demands and important social significance.

The pig plays a significant role in the market of China as the main meat source of China. Under the current background of large-scale breeding, the pig viral diseases always puzzle and threaten the development of the pig industry. Interferon (IFN) plays an important role in swine anti-infective immunity as a highly effective non-specific immune factor. Type III interferons, i.e., lambda interferon (IFN-. lambda.), are cytokines classified as type III interferons that were simultaneously discovered in vertebrates by two independent research groups in 2003 and are classified as members of the IL-10 family due to their genetic structure similarity to IL-10. Type III interferons include IFN-. lambda.1 (IL-29), IFN-. lambda.2 (IL-28. alpha.), and IFN-. lambda.3 (IL-28. beta.). The III type interferon lambda has broad-spectrum antiviral capability, compared with the I type interferon, the receptor of the III type interferon lambda is only limited and distributed in epithelial cells and certain dendritic cell subgroups, the effect of the III type interferon lambda has obvious tissue specificity and small toxic and side effects, and the III type interferon lambda has the potential of being applied to clinical antiviral treatment. At present, the porcine interferon gene sequence and the amino acid sequence are reported at home and abroad, the expression of the interferon gene in a prokaryotic or eukaryotic expression system is realized, a basis is provided for the large-scale production and application of porcine genetic engineering interferon, and the problems of low expression level, low antiviral activity, low yield and the like still exist, so that the porcine interferon gene is further optimized to improve the expression level of the porcine interferon at present, a more efficient protein expression system is developed, and the method has important economic significance and industrial application prospect.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the expression level of the recombinant porcine lambda 3 interferon by optimizing the gene of the encoded porcine lambda 3 interferon. The technical problem to be solved is not limited to the technical subject as described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

In order to solve the technical problems, the invention firstly provides a method for preparing the recombinant porcine lambda 3 interferon, which comprises the steps of introducing a DNA molecule into escherichia coli to obtain the recombinant escherichia coli; culturing the recombinant Escherichia coli, and performing induced expression to obtain the recombinant porcine lambda 3 interferon, wherein the nucleotide sequence of the DNA molecule is SEQ ID No.1 or SEQ ID No. 4.

Further, the method can be that recombinant plasmids containing optimized DNA molecules are introduced into escherichia coli to obtain recombinant escherichia coli; culturing the recombinant Escherichia coli, and performing induction expression to obtain the recombinant porcine lambda 3 interferon, wherein the nucleotide sequence of the DNA molecule can be SEQ ID No.1 or SEQ ID No. 4.

Further, the invention provides a method for efficiently preparing recombinant porcine lambda 3 interferon, which comprises the steps of introducing a recombinant plasmid containing an optimized DNA molecule (SEQ ID No.1 or SEQ ID No.4) into escherichia coli to obtain recombinant escherichia coli; culturing the recombinant escherichia coli, and performing induction expression to obtain the recombinant porcine lambda 3 interferon.

The DNA molecule may be a codon optimized porcine lambda 3 interferon gene (PoIFN lambda 3-MD); the PoIFN lambda 3-MD nucleotide sequence is SEQ ID No. 1;

the DNA molecule can be a fusion gene of an optimized porcine lambda 3 interferon gene (PoIFN lambda 3-MD) and a tag protein;

further, the tag protein may be a His-tag protein;

the fusion gene can be a fusion gene His-PoIFN lambda 3-MD, and the nucleotide sequence of the fusion gene His-PoIFN lambda 3-MD is SEQ ID No. 4.

The invention also provides a protein coded by the optimized pig lambda 3 interferon gene, and the amino acid sequence of the protein is SEQ ID No. 2.

The invention also provides a protein coded by the fusion gene His-PoIFN lambda 3-MD, and the amino acid sequence of the protein is SEQ ID No. 5.

Further, in the above method, the Escherichia coli may be Escherichia coli BL21(DE 3);

further, the induced expression can be induced by 0.5mmol/L IPTG liquid at 16-37 ℃ for 4-16 hours, such as 0.5mmol/L IPTG liquid at 37 ℃ for 6 hours.

In the above method, the method may specifically include the steps of:

(1) introducing the DNA molecule PoIFN lambda 3-MD (SEQ ID No.1) into escherichia coli to obtain recombinant escherichia coli (BL21pET30a-PoIFN lambda 3-MD);

(2) adding 0.5mmol/L IPTG (isopropyl-beta-D-thiogalactoside) into the recombinant escherichia coli, inducing for 4-16 hours at 16-37 ℃, then ultrasonically crushing thalli, collecting inclusion bodies, washing the inclusion bodies, dissolving the inclusion bodies, purifying and then renaturing the inclusion bodies to obtain a solution containing the recombinant porcine lambda 3 interferon.

In the above method, the method further comprises a step of purifying the recombinant porcine lambda 3 interferon.

The invention also provides a biomaterial, which can be any one of the following:

A1) an expression cassette containing the DNA molecule PoIFN lambda 3-MD or His-PoIFN lambda 3-MD;

A2) a recombinant vector containing the DNA molecule PoIFN lambda 3-MD or His-PoIFN lambda 3-MD, or a recombinant vector containing A1) the expression cassette;

A3) a recombinant microorganism containing the DNA molecule PoIFN lambda 3-MD or His-PoIFN lambda 3-MD, or a recombinant microorganism containing A1) the expression cassette, or a recombinant microorganism containing A2) the recombinant vector;

A4) a transgenic cell line containing the DNA molecule PoIFN lambda 3-MD or His-PoIFN lambda 3-MD, or a transgenic cell line containing A1) the expression cassette, or a transgenic cell line containing A2) the recombinant vector.

The recombinant vector can be a recombinant vector obtained by inserting the DNA molecule PoIFN lambda 3-MD shown in SEQ ID No.1 into the multiple cloning site of the vector pET30a (+) (namely, the recombinant plasmid pET30a-PoIFN lambda 3-MD).

Further, the recombinant vector (i.e., recombinant plasmid pET30 a-PoIFN. lamda.3-MD) may be a recombinant expression vector obtained by replacing the fragment between the Xho I recognition site and the EcoR V recognition site of pET30a (+) (a small fragment between the Xho I recognition site and the EcoRV recognition site) with the DNA molecule PoIFN. lamda.3-MD shown in SEQ ID No.1, while keeping the other sequence of pET30a (+) unchanged.

The recombinant bacterium can be obtained by introducing a DNA molecule PoIFN lambda 3-MD shown in SEQ ID No.1 into escherichia coli BL21(DE 3);

the recombinant bacteria can be obtained by introducing a DNA molecule His-PoIFN lambda 3-MD shown in SEQ ID No.4 into escherichia coli BL21(DE 3);

further, the recombinant bacterium can be a recombinant bacterium obtained by introducing the recombinant vector (i.e., the recombinant plasmid pET30a-PoIFN lambda 3-MD) into Escherichia coli BL21(DE3), which is named as Escherichia coli (Escherichia coli) BL21(DE3) pET30a-PoIFN lambda 3-MD (BL21 (DE3) pET30a-PoIFN lambda 3-MD for short).

The invention also provides a method for preparing the recombinant porcine lambda 3 interferon, which comprises the step of preparing the recombinant porcine lambda 3 interferon by using the recombinant microorganism or the transgenic cell line.

DNA molecules described herein are also within the scope of the invention.

Recombinant porcine lambda 3 interferon produced by any of the methods described herein is also within the scope of the present invention.

The invention also provides the application of the DNA molecule PoIFN lambda 3-MD or His-PoIFN lambda 3-MD and/or the biological material in preparing a medicine or a preparation for treating and/or preventing porcine viral diseases.

In the above application, the preparation may be an antiviral inhibitor.

The invention also provides the DNA molecule PoIFN lambda 3-MD or His-PoIFN lambda 3-MD, and/or the application of the biological material in the preparation of recombinant porcine lambda 3 interferon.

The invention provides an optimized gene sequence for coding PoIFN lambda 3-MD, wherein the expression capacity of the gene in a prokaryotic expression system is far higher than that of an unoptimized gene. The gene is inserted into a multiple cloning site of a vector pET30a (+) to obtain a recombinant plasmid, and the recombinant plasmid is introduced into escherichia coli BL21(DE3) to obtain a recombinant bacterium. The recombinant strain can prepare a large amount of recombinant porcine lambda 3 interferon protein by simple induction culture. The gene, the recombinant plasmid and the recombinant bacterium provided by the invention have great economic value for the production of the recombinant porcine lambda 3 interferon and also have great value for the live pig breeding industry.

Drawings

FIG. 1 shows the sequence alignment of PoIFN lambda 3-MD gene (DNA shown in SEQ ID No.1) and PoIFN lambda 3-WT gene (DNA shown in SEQ ID No. 3).

FIG. 2 shows the results of SDS-PAGE detecting the expression products of porcine interferon genes (PoIFN. lambda.3-MD and PoIFN. lambda.3-WT) in E.coli; UI stands for non-induced, I stands for induced, and M is a low molecular weight protein Marker.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

All primers and gene synthesis and sequencing work in the examples described below was performed by Biotechnology engineering (Shanghai) Inc. and Suzhou Jinzhi Biotechnology Inc.

The vector pET30a (+) from the following examples was purchased from Addgene, cat # 85761.

Example 1 Artificial Synthesis of optimized porcine Lambda 3 Interferon Gene and control Gene

Artificial synthesis of optimized pig lambda 3 interferon gene (PoIFN lambda 3-MD gene)

According to the degeneracy of amino acid codons and the codon preference of Escherichia coli, a coding Gene of the porcine lambda 3 interferon containing the codon preference of Escherichia coli is designed by referring to a porcine lambda 3 interferon Gene sequence (Gene ID: 100310828) in Genebank. The nucleotide sequence of the optimized encoding pig lambda 3 interferon gene (PoIFN lambda 3-MD gene) is shown in SEQ ID No.1 and contains 519 nucleotides in total.

The amino acid sequence of the protein coded by the optimized pig lambda 3 interferon gene shown in SEQ ID No.1 is SEQ ID No. 2.

Cloning of control Gene (PoIFN. lambda.3-WT Gene)

The control Gene, i.e., the pig λ 3 interferon Gene in Genebank, was amplified by designing primers with reference to the pig λ 3 interferon Gene sequence in Genebank (Gene ID: 100310828). The nucleotide sequence of the control gene (PoIFN lambda 3-WT) is shown in SEQ ID No.3, and the control gene contains 519 nucleotides in total.

Alignment of PoIFN lambda 3-MD Gene and PoIFN lambda 3-WT Gene

The sequence alignment of PoIFN lambda 3-MD gene and PoIFN lambda 3-WT gene is shown in figure 1, and both PoIFN lambda 3-MD gene and PoIFN lambda 3-WT gene encode recombinant porcine lambda 3 interferon shown in SEQ ID No. 2.

Example 2 construction of recombinant plasmid and recombinant bacterium and expression of Gene

Construction of recombinant plasmid and engineering bacterium containing PoIFN lambda 3-MD gene

The fragment between the Xho I recognition site and the EcoR V recognition site of pET30a (+) (a small fragment between the Xho I recognition site and the EcoR V recognition site) was replaced with a DNA molecule (PoIFN. lamda.3-MD gene) whose nucleotide sequence was SEQ ID No.1, and the other sequence of pET30a (+) was kept unchanged to obtain a recombinant vector pET30 a-PoIFN. lamda.3-MD. The sequence of the sequence to be detected is analyzed by the SnapGene software to be consistent with the optimized sequence by the company of biological engineering (Shanghai). pET30a-PoIFN lambda 3-MD contains His-PoIFN lambda 3-MD fusion gene with the nucleotide sequence of SEQ ID No.4, the fusion gene is a section of gene fused with His label and carrier, the coding sequence of His-PoIFN lambda 3-MD gene is SEQ ID No.4, and the amino acid sequence coded by His-PoIFN lambda 3-MD gene is protein His-PoIFN lambda 3-MD with SEQ ID No. 5. Positions 48-219 of SEQ ID No.5 are the amino acid sequence of the protein PoIFN λ 3-MD (i.e., SEQ ID No. 2). The nucleotide sequence of PoIFN lambda 3-MD gene (i.e., SEQ ID No.1) is located at position 142-660 of SEQ ID No. 4.

The recombinant plasmid pET30a-PoIFN lambda 3-MD is transformed into escherichia coli BL21(DE3) to obtain recombinant bacteria BL21(DE3) pET30a-PoIFN lambda 3-MD.

Secondly, expression of PoIFN lambda 3-MD gene:

the overnight-cultured BL21(DE3) pET30a-PoIFN lambda 3-MD was transferred to 400ml LB medium containing kanamycin (kanamycin concentration 0.5ug/ml), cultured with shaking at 37 ℃ and 220r/min for 3 hours (to bacterial solution OD600 ═ 1), added with 0.5mmol/L IPTG, and induced with shaking at 37 ℃ and 220r/min for 6 hours to obtain BL21(DE3) pET30a-PoIFN lambda 3-MD fermentation broth (post-induction culture). 1ml of culture solution before and after induction is respectively taken out and put in a centrifuge tube, 12000r/min is carried out, centrifugation is carried out for 10 minutes at 4 ℃, and the supernatant is discarded to collect the precipitate. After adding 6 XLoading Buffer and boiling for 10 minutes at 100 ℃, SDS-PAGE electrophoresis detection is carried out. The results of electrophoresis are shown in FIG. 2, and a protein band appeared around 26kD in the collected thallus after induction, which is consistent with the expected size.

Thirdly, denaturation, purification and renaturation of recombinant porcine lambda 3 interferon protein

1. Denaturation of the material

After the thalli expressed by IPTG induction are broken by ultrasonic (phi 6 probe, ultrasonic 4 seconds interval 5 seconds, 30 minutes), the inclusion body precipitate is collected, the weight of the thalli collected by engineering bacteria is 0.78g, and the weight of a control group is 0.75 g. And (3) adding 20ml of solution into each gram of bacteria, washing the inclusion body precipitate for 2-3 times by using the solution A, and stirring overnight at 4 ℃ by using the solution B to fully dissolve the inclusion body protein. The pH value of the solution A is 8.0, the solution A consists of urea, Tris-HCl, EDTA, NaCl, beta-mercaptoethanol, Triton-100 and water, the concentration of the urea is 2mol/L, the concentration of the Tris-HCl is 50mmol/L, EDTA, the concentration of the Tris-L, NaCl is 50mmol/L, and the concentration of the beta-mercaptoethanol is 2mmol/L, Triton-100, and the concentration of the beta-mercaptoethanol is 1%; the pH value of the solution B is 8.0, the solution B is composed of urea, Tris-HCl, beta-mercaptoethanol and water, the concentration of the urea is 8mol/L, the concentration of the Tris-HCl is 50mmol/L, and the concentration of the beta-mercaptoethanol is 2 mmol/L.

2. Purification of

Centrifuging the denatured solution at 12000r/min at 4 deg.C for 10min, collecting supernatant, filtering with 0.45 μm filter membrane, and purifying with Ni-NTA affinity chromatography column. Performing affinity chromatography column purification on a solution containing the recombinant porcine lambda 3 interferon by using a GE AKTA Pure protein separation and purification system, using a binding buffer solution to bind a target protein at the flow rate of 0.5ml/min, then washing 10 column volumes by using a washing buffer solution, finally washing 5-10 column volumes by using an elution buffer solution, and collecting an eluent with the ultraviolet absorption value of more than 200 under 280nm illumination to obtain a recombinant porcine lambda 3 interferon solution; the pH value of the binding buffer solution is 8.0, the binding buffer solution consists of Tris-HCl, imidazole, urea and water, the concentration of the Tris-HCl is 50mmol/L, the concentration of the imidazole is 10mmol/L, and the concentration of the urea is 8M; the pH value of the impurity washing buffer solution is 8.0, the impurity washing buffer solution consists of Tris-HCl, imidazole, urea and water, the concentration of the Tris-HCl is 50mmol/L, the concentration of the imidazole is 80mmol/L, and the concentration of the urea is 8M; the pH value of the elution buffer solution is 8.0, the elution buffer solution is composed of Tris-HCl, imidazole, urea and water, the concentration of the Tris-HCl is 50mmol/L, the concentration of the imidazole is 500mmol/L, and the concentration of the urea is 8M.

3. Renaturation

And dialyzing the purified protein in a renaturation buffer solution with descending urea concentration gradient, changing the renaturation buffer solution every 3 hours, and finally dialyzing in a PBS buffer solution for 12-24 hours. Placing the dialysis bag into a large plate, concentrating with PEG8000, filtering the concentrated protein for sterilization, measuring concentration, and storing at-80 deg.C. The pH value of the urea renaturation buffer solution is 8.0, the buffer solution comprises urea, Tris-HCl, NaCl, L-arginine, oxidized glutathione, reduced glutathione and water, the urea concentration is 6mol/L, 4mol/L, 2mol/L and 0mol/L in a gradient descending manner, the Tris-HCl concentration is 50mmol/L, the NaCl concentration is 0.15mol/L, the L-arginine concentration is 0.5mol/L, the oxidized glutathione concentration is 0.4mmol/L, and the reduced glutathione concentration is 2 mmol/L.

And (3) obtaining a solution (PoIFN lambda 3-MD solution and PoIFN lambda 3-WT solution) containing the recombinant porcine lambda 3 interferon through the steps 1, 2 and 3.

Fourthly, determining the protein amount of the optimized gene sequence PoIFN lambda 3-MD in the host bacteria

The solution to be detected is PoIFN lambda 3-MD solution or PoIFN lambda 3-WT solution obtained in the third step.

Taking solution A and solution B in a BCA protein quantitative kit (purchased from Shanghai Solibao biotechnology Co., Ltd., product number: PC0020) according to the volume ratio of 50:1 to mix into working solution, diluting BSA standard substance to 500mg/ml, 400mg/ml, 300mg/ml, 200mg/ml, 150mg/ml, 100mg/ml and 50mg/ml in a gradient manner, taking 20 mu l of standard substance or solution to be detected of each concentration, adding 200 mu l of working solution to mix, sealing by using a preservative film, incubating for 30min at 37 ℃, recovering to room temperature, and reading the absorbance at 562nm by using an enzyme reader. And drawing a standard curve according to the concentration and the light absorption value of the standard substance, and calculating the protein concentration in the protein sample to be detected according to the standard curve.

The results showed that from 400mL of BL21(DE3) pET30a-PoIFN λ 3-MD fermentation broth (post-induction broth, OD600nm ═ 1.4), 3mL of PoIFN λ 3-MD solution was purified according to the above procedure with a protein concentration of 0.6mg/mL, yielding 1.8mg of recombinant protein altogether; in contrast, from 400mL of BL21(DE3) pET30a-PoIFN λ 3-WT broth (post-induction broth OD600nm ═ 1.4), 3mL of PoIFN λ 3-WT solution was purified according to the above procedure to a protein concentration of 0.3mg/mL, and 0.9mg of recombinant protein was co-harvested. The result shows that the expression capacity of the optimized gene PoIFN lambda 3-MD in host bacteria is far higher than that of the unoptimized gene PoIFN lambda 3-WT.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

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Claims

1. A method for preparing recombinant porcine lambda 3 interferon is characterized by comprising the steps of introducing a DNA molecule into Escherichia coli to obtain recombinant Escherichia coli; culturing the recombinant Escherichia coli, and performing induced expression to obtain the recombinant porcine lambda 3 interferon, wherein the nucleotide sequence of the DNA molecule is SEQ ID No.1 or SEQ ID No. 4.

2. The method of claim 1, further comprising the step of purifying the recombinant porcine lambda 3 interferon.

3. A biomaterial, characterized in that the biomaterial is any one of the following:

A1) an expression cassette comprising the DNA molecule of claim 1;

A2) a recombinant vector comprising the DNA molecule of claim 1, or a recombinant vector comprising a1) the expression cassette;

A3) a recombinant microorganism comprising the DNA molecule of claim 1, or a recombinant microorganism comprising A1) the expression cassette, or a recombinant microorganism comprising A2) the recombinant vector;

A4) a transgenic cell line comprising the DNA molecule of claim 1, or a transgenic cell line comprising A1) the expression cassette, or a transgenic cell line comprising A2) the recombinant vector.

4. The DNA molecule of claim 1.

5. A recombinant porcine lambda 3 interferon produced by the method of claim 1 or 2.

6. Use of a DNA molecule as claimed in claim 1, and/or a biomaterial as claimed in claim 3 in the manufacture of a medicament or formulation for the treatment and/or prevention of porcine viral disease.

7. The use of claim 6, wherein the agent is an antiviral inhibitor.

8. The use according to claim 6, wherein the formulation is a formulation for improving survival of piglets.

9. The use according to any one of claims 6 to 8, wherein the porcine viral disease is porcine epidemic diarrhea, porcine vesicular stomatitis, transmissible gastroenteritis, rotavirus disease, porcine circovirus disease, porcine ear disease, porcine foot and mouth disease, swine fever or porcine viral cold.

10. Use of the DNA molecule of claim 1, and/or the biomaterial of claim 3 in the preparation of recombinant porcine λ 3 interferon.