阅读说明：本技术 一种猪干扰素-α8突变体及其制备方法 (Porcine interferon-alpha 8 mutant and preparation method thereof ) 是由 付伟 高小平 代燕平 李晟 罗弟祥 于 2020-05-26 设计创作，主要内容包括：本发明公开了一种猪干扰素-α8(PoIFN-α8)突变体及其制备方法。该突变体具有(1)在第18位上将丙氨酸由脯氨酸取代和第21位上将丝氨酸由缬氨酸取代；(2)在101位点上将Asp置换为Asn,形成N-连接型糖蛋白的Asn-X-Ser糖基化修饰；(2)本发明还涉及PoIFN-α8突变体的应用,注射给药后,可显著增加PoIFN-α8突变体的血药浓度-时间曲线下面积。(The invention discloses a porcine interferon-alpha 8 (PoIFN-alpha 8) mutant and a preparation method thereof. The mutant has (1) an alanine substituted with proline at position 18 and a serine substituted with valine at position 21; (2) the Asp is replaced by Asn at the 101 site to form Asn-X-Ser glycosylation modification of the N-linked glycoprotein; (2) the invention also relates to application of the PoIFN-alpha 8 mutant, and after injection administration, the area under a blood concentration-time curve of the PoIFN-alpha 8 mutant can be obviously increased.)

1. A porcine interferon- α 8 mutant (PoIFN- α 8), which is characterized in that the PoIFN- α 8 mutant has an alanine (Ala) at position 18 replaced by proline (Pro), a serine (Ser) at position 21 replaced by valine (Val), an aspartic acid (Asp) at position 101 replaced by asparagine (Asn), and a carboxyl terminal added with 6 × histidine (His), and has an amino acid sequence shown in SEQ ID NO 1.

2. A nucleotide sequence encoding the PoIFN- α 8 mutant of claim 1, as shown in SEQ ID NO 2.

3. The method of preparing PoIFN- α 8 mutants of claim 1, comprising the steps of:

(1) artificially synthesizing a nucleotide sequence shown as SEQ ID NO2, and introducing restriction enzymes Avr II and Pac I sites at two ends of the nucleotide sequence;

(2) carrying out double enzyme digestion by using restriction enzymes Avr II and Pac I, inserting the double enzyme digestion into a plasmid pUCEE-M with an ampicillin resistance marker, and constructing a recombinant PoIFN-alpha 8 (rPoIFN-alpha 8) mutant expression plasmid;

(3) electrically transducing the constructed expression plasmid into a host CHO cell line, preferably CHO-DG44 cell, culturing for 48h in serum-free medium, adding Methotrexate (MTX) to a final concentration of 10nM, and gradually increasing to 100-500 nM; screening to obtain an engineering cell line expressing the PoIFN-alpha 8 mutant.

4. The rpoIFN- α 8 mutant according to claims 1-3 for use in a medicament for the treatment of porcine viral infectious disease.

Technical Field

The invention relates to the technical field of biological veterinary medicine, and in particular relates to a PoIFN-alpha 8 mutant and a preparation method thereof.

Background

Interferons (IFNs) bind to their receptors and induce cells to produce antiviral proteins with enzymatic activity, thereby playing roles in antiviral and immunoregulation, and the like. The antiviral activity induced by IFN has species difference and subtype difference, porcine interferon (PoIFN) is mainly divided into type I, type II and type III, the type I comprises IFN-alpha, beta, delta, omega and tau, the IFN-gamma is type II, the IFN-lambda belongs to type III, the porcine interferon type I has the strongest antiviral activity, and the IFN-alpha has the widest antiviral effect and the most obvious effect. The recombinant PoIFN-alpha 1 has good biological activity and is widely used in antiviral preparations, and PoIFN-alpha 8 also has high antiviral activity and is obviously stronger than IFN-alpha 1 in partial virus effects such as vesicular stomatitis resistance, so that PoIFN-alpha 8 also has extremely important clinical application value.

Both the recombinant IFN-alpha and the natural IFN-alpha obtained by separation have the limitations of short half-life and fast clearance, and the antiviral activity of the recombinant IFN-alpha (rIFN-alpha) is still not ideal. Chinese patent (CN104818283B) discloses an optimized PoIFN-alpha 8 gene and an expression method thereof, although PoIFN-alpha 8 is expressed in a prokaryotic form in a soluble manner, PoIFN-alpha 8 expressed in the prokaryotic form cannot be subjected to glycosylation modification. The aglycosylated modified interferon has limited clinical use because of its short half-life and the need for frequent administration over a long period of time. Therefore, increasing the half-life of interferons is a direction of development in the therapeutic field. Currently, interferons that extend half-life are predominantly in PEGylated form, e.g.Andhas been clinically used for the treatment of hepatitis B and hepatitis C. In addition, the introduction or substitution of N-glycosylation modification sites in the interferon molecule is also an effective means for extending the plasma half-life of recombinant interferon. If 4-5N-glycosylation modifications (Ceaglio, N, etc.; 2008) are introduced into human interferon-a (huIFN-a), the plasma half-life can be significantly prolonged by subcutaneous injection and compared with non-N-glycosylation modified huIFN-a.

In order to prolong the plasma half-life of PoIFN-alpha 8, the invention aims at the Asp-X-Ser sequence of the PoIFN-alpha 8 molecule, wherein Asp (position 101) is replaced by Asn, and N-linked Asn-X-Ser glycosylation modification is formed. In addition, the inventor of the patent discovers that PoIFN-alpha 8 carrying the self signal peptide can be successfully expressed and secreted in the CHO cell by applying a CHO cell expression system, but the enzyme cutting specificity of the self signal peptide is poor, so that the expression yield of the PoIFN-alpha 8 can not meet the industrialization requirement. Therefore, the signal peptide of PoIFN-alpha 8 is further mutated, so that PoIFN-alpha 8 is efficiently expressed and secreted in CHO cells.

Disclosure of Invention

The present invention provides a PoIFN- α 8 mutant in which alanine (Ala) at position 18 is replaced with proline (Pro), serine (Ser) at position 21 is replaced with valine (Val), aspartic acid (Asp) at position 101 is replaced with asparagine (Asn), and 6 × histidine (His) is added at the carboxyl terminal.

The invention provides an amino acid sequence of a PoIFN-alpha 8 mutant shown as SEQ ID NO. 1.

In order to obtain the PoIFN-alpha 8 mutant provided by the invention, the invention provides a preparation method of the PoIFN-alpha 8 mutant, which comprises the following steps:

(1) artificially synthesizing PoIFN-alpha 8 mutant nucleotide sequences, respectively introducing endonuclease sites Avr II and Pac I at two ends of the nucleotide sequences, introducing a GCCACC sequence before ATG, and adding a 6 XHis sequence at the tail end, wherein the sequence is shown as SEQ ID NO. 2. Connecting the artificially synthesized PoIFN-alpha 8 mutant nucleotide sequence with a UCOE expression vector (pUCEE-M) optimized for modification, transforming escherichia coli, and constructing a pUCEE-M/rPoIFN-alpha 8M expression plasmid through expression, screening and double enzyme digestion identification.

(2) Linearizing a pUCOE-M/rPoIFN-alpha 8M expression plasmid, respectively electrically transferring the plasmid to CHO cells, preferably CHO DG44 cells, culturing for 48h in a serum-free manner, adding Methotrexate (MTX) to a final concentration of 10nM, and then gradually increasing to 100-500 nM;

(3) cells with increasing MTX to 500nM were inoculated at the same density into shake flasks, continuously cultured in commercial medium and appropriately supplemented with nutritional supplements such as glucose. On day 12 of continuous culture, the supernatant was collected, and the amount of interferon secreted in the supernatant was quantitatively determined by ELISA.

The invention has the beneficial effects that:

compared with the prior art, on one hand, the 101-site Asp is replaced by Asn to form an Asn-X-Ser sequence, so that the invention realizes glycosylation modification in mammalian cells and prolongs the half-life of the PoIFN-alpha 8 in plasma. On the other hand, the invention replaces Ala at the 18 th position with Pro and Ser at the 21 st position with Val, the cleavage specificity of the signal peptide at the 21 st position is lost, the cleavage specificity of the signal peptide at the 23 rd position is improved, and the high-efficiency expression and secretion of the PoIFN-alpha 8 mutant in mammalian cells are realized.

Drawings

The drawings are included to provide a further understanding and explanation of the invention and are not to be construed as limiting the invention.

FIG. 1 shows the identification of rPoIFN-alpha 8m plasmid target gene enzyme digestion product

FIG. 2 shows rPoIFN-. alpha.8m protein expression levels

FIG. 3 shows the molecular weight of purified rPoIFN-. alpha.8M

Detailed Description

Example 1 rPoIFN-. alpha.8 mutant expression plasmid construction and characterization

The PoIFN-. alpha.8 sequence (accession No.: GQ415062.1) was obtained from GenBank and its amino acid sequence is shown in SEQ ID NO 3. The codon encoding alanine at position 18 is mutated to CCG, the codon encoding serine at position 21 is mutated to GTG, the codon encoding aspartic acid at position 101 is mutated to AAT, and codon preference optimization is performed. Respectively introducing endonuclease sites Avr II and Pac I at the 5 'end and the 3' end, introducing a GCCACC sequence after the Avr II endonuclease site and before ATG, and adding a 6 × His sequence at the tail end; artificially synthesizing PoIFN-alpha 8 mutant (PoIFN-alpha 8M) gene, connecting the PoIFN-alpha 8 mutant gene with UCOE expression vector (pUCEE-M) optimized by modification, transforming Top10 competent cells, and obtaining rPoIFN-alpha 8M expression plasmid; the expression plasmid was double digested with Avr II and Pac I restriction enzymes, and the plasmid digested products were identified by 1% agarose gel (FIG. 1), showing that the rPoIFN-. alpha.8M expression plasmid was successfully constructed and the rPoIFN-. alpha.8 expression plasmid was constructed at the same time.

Example 2 plasmid transfection and protein expression

The rPoIFN-alpha 8M and rPoIFN-alpha 8 expression plasmids are extracted by adopting an Axygen Plasmid Max Kit, after linearization is carried out by Pvu I endonuclease, the linearized rPoIFN-alpha 8M and rPoIFN-alpha 8 plasmids are stably transfected to CHO DG44 cells respectively by referring to an Amaxa Cell Line nucleofector Kit V Kit description method. After the transfected cells were cultured for 48 hours in serum-free medium, the culture was continued by adding each of the selection media containing 10nM MTX, followed by stepwise increase of the MTX concentration from 100nM to 500 nM.

Cells with increased MTX to 500nM were inoculated at the same density into shake flasks and cultured continuously for 12 days in commercial medium Acti Pro (cell density 5X 10 each)⁵cells/ml, 50ml volume), and 5% Cell Boost 7a/7b was added on days 3, 6, 8, and 10 of the culture, and 1% glucose and 1% L-Glutamine were added in appropriate amounts. The culture was terminated on the 12 th day of culture and supernatants from each culture were collected separately, and rPoIFN-. alpha.was quantified by ELISA. The results show that the signal peptide mutation leads to a significantly higher expression yield of rPoIFN-. alpha.8m and increases with time, while the production yield of rPoIFN-. alpha.8 with its own signal peptide also increases with time, but is significantly lower than the expression of rPoIFN-. alpha.8m (FIG. 2).

Example 3 purification of recombinant PoIFN-. alpha.8m

Collecting cell supernatant cultured on day 12, filtering, and purifying protein by Ni column affinity chromatography to obtain rPoIFN-alpha 8m sample, and subjecting the purified rPoIFN-alpha 8m to non-reduction and reduction SDS-PAGE electrophoresis. According to the electrophoresis result, the molecular weight of rPoIFN-alpha 8m expressed by the recombinant cells is consistent with the theoretical expectation and is about 20kDa (figure 3), which indicates that the rPoIFN-alpha 8m is expressed correctly.

Example 4 in vivo half-life of rPoIFN-. alpha.8m of the invention in rats

16 SD rats with the weight of 220-250 g are divided into two groups, each group comprises 8 SD rats, and purified rPoIFN-alpha 8m and rPoIFN-alpha 8 samples are respectively injected into the SD rats through intravenous injection, and the injection dose is 5 mg/kg. Blood is collected at orbital veins of 0min, 5min, 10min, 15min, 30min, 1h, 2h, 4h, 8h, 16h, 24h, 48h and 96h after injection, the sample is placed at room temperature for 30min, serum is separated by low-temperature centrifugation at 3500rpm for 10min, and the rPoIFN-alpha 8m content in the blood is detected by ELISA. DAS pharmacokinetic software was used for curve fitting and parameter calculation, and the results show: compared with rPoIFN-alpha 8, the half-life period of rPoIFN-alpha 8m provided by the invention is obviously increased, and the blood content is obviously increased (Table 1).

TABLE 1 major kinetic parameters in serum after rPoIFN-. alpha.8 m intravenous injection

Sequence listing

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