阅读说明：本技术 一种重组马血清白蛋白及其制备方法和应用 (Recombinant horse serum albumin and preparation method and application thereof ) 是由 范铁炯 胡金贵 胡双锋 孙九如 梁光军 史小月 柏伟 李鑫 于 2019-12-12 设计创作，主要内容包括：本发明公开了一种重组马血清白蛋白及其制备方法,将经过密码子优化的编码马血清白蛋白的核苷酸序列(如SEQ ID NO:2所示)构建入真核细胞诱导表达载体中,然后转入真核细胞进行培养后诱导表达,并进行纯化,从而获得所述重组马血清白蛋白。此外本发明还公开了该重组马血清白蛋白在制备免疫马匹用的狂犬病疫苗中的应用。用本发明重组马血清白蛋白作为稳定剂的狂犬病抗原免疫马匹后获得高抗狂犬病毒抗体,未检测出抗人血清白蛋白的抗体。本发明获得的重组马血清白蛋白可以替代人血清白蛋白作为免疫马匹用狂犬病疫苗的稳定剂,用于生产高质量马抗人狂犬病毒血清。(The invention discloses a recombinant horse serum albumin and a preparation method thereof, wherein a nucleotide sequence (shown as SEQ ID NO: 2) which is optimized by a codon and used for coding the horse serum albumin is constructed into an eukaryotic cell induction expression vector, then is transferred into eukaryotic cells for culture, then is subjected to induction expression, and is purified, so that the recombinant horse serum albumin is obtained. In addition, the invention also discloses application of the recombinant horse serum albumin in preparing rabies vaccines for immunizing horses. The high anti-rabies virus antibody is obtained after the horse is immunized by the rabies antigen taking the recombinant horse serum albumin as the stabilizer, and the anti-human serum albumin antibody is not detected. The recombinant horse serum albumin obtained by the invention can replace human serum albumin to be used as a stabilizer of a rabies vaccine for immune horses, and is used for producing high-quality horse anti-human rabies virus serum.)

1. A preparation method of recombinant horse serum albumin is characterized in that a nucleotide sequence which is optimized by codon and used for coding the horse serum albumin is constructed into an eukaryotic cell induced expression vector, then the vector is transferred into the eukaryotic cell for culture, induced expression is carried out, and purification is carried out, so as to obtain the recombinant horse serum albumin; the nucleotide sequence of the coded horse serum albumin which is optimized by the codon is a sequence shown as SEQ ID NO. 2.

2. The method of claim 1, wherein the culturing comprises a preliminary step of pre-seeding.

3. The method of claim 1, wherein the eukaryotic cell is a yeast cell.

4. The method of claim 3, wherein the cell is a Pichia pastoris cell.

5. The method of claim 4, wherein the Pichia pastoris cell is Pichia pastoris GS 115.

6. The method of claim 1, wherein said eukaryotic cell inducible expression vector is a secretory expression vector, and after said inducible expression, cells and particulate matter are removed by centrifugation, and the supernatant is removed and subjected to said purification.

7. The method for preparing recombinant horse serum albumin according to claim 6, characterized in that the secretion expression vector is a Pichia pastoris expression vector, in particular a pHIL-D2 expression vector, and contains a horse serum albumin expression cassette, and the secretion signal peptide is a horse serum albumin self-secretion signal peptide.

8. The method for preparing recombinant horse serum albumin according to claim 7, characterized in that the codon optimized nucleotide sequence coding horse serum albumin is optimized according to the codon preferred by yeast, EcoR I is added before the initiator and after the terminator, cDNA expression cassettes are artificially synthesized and inserted into EcoRI site of Pichia pastoris expression vector pHIL-D2 to construct ESA horse serum albumin/pHIL-D2 expression vector; taking a certain amount of pHIL-D2 plasmid DNA, carrying out enzyme digestion linearization by SalI, adding into Pichia pastoris culture, transferring the plasmid DNA into yeast by an electric shock method, integrating the plasmid DNA into yeast genome DNA, and screening by using a histidine-deficient culture medium to obtain a cell strain for expressing albumin.

9. The method of claim 1, wherein the transfer into eukaryotic cells is performed to induce expression after culturing in YPD/YPM medium comprising 1% yeast extract, 2% peptone, and 2% glucose or 0.5% methanol.

10. The method for preparing recombinant horse serum albumin according to claim 9, wherein the culturing step is specifically: selecting clone, inoculating into YPD culture medium, culturing at 30 deg.C and 200rpm for 24-72 hr; centrifuging to remove supernatant, changing culture medium to YPM, culturing at 28 deg.C and 200rpm for 24-120 hr.

11. The method for preparing recombinant horse serum albumin according to claim 10, wherein the culturing step is specifically: selecting clone, inoculating into YPD culture medium, culturing at 30 deg.C and 200rpm for 48 hr; centrifuging to remove supernatant, changing culture medium to YPM, culturing at 28 deg.C and 200rpm for 48-96 hr.

12. The method for preparing recombinant horse serum albumin according to claim 11, wherein the culturing step is specifically: selecting clone, inoculating into YPD culture medium, culturing at 30 deg.C and 200rpm for 48 hr; the supernatant was centrifuged off, YPM was used as a medium, and the medium was cultured at 28 ℃ and 200rpm for 72 hours.

13. The method of claim 1 or 7, wherein said inducible expression is achieved by stepwise addition of methanol during yeast culture, with methanol being added at intervals of 0.5-1.5% every 24 hours.

14. The method of claim 13, wherein 1% methanol is added every 24 hours.

15. The method for producing recombinant horse serum albumin according to claim 6, wherein the purification method comprises separating and purifying the culture supernatant obtained by centrifugation by anion-cation exchange chromatography; separating and purifying by anion-cation exchange chromatography, and adopting cation exchange gel CM-Sepharose FF gel or SP-Sepharose FF gel; anion exchange gel Q-Sepharose FF gel or DEAE-Sepharose FF gel is used.

16. The method of claim 15, wherein the cation exchange gel is an SP-Sepharose FF gel; the anion exchange gel is DEAE-Sepharose FF gel.

17. A recombinant horse serum albumin produced by the production method according to any one of claims 1 to 16.

18. Use of recombinant horse serum albumin according to claim 17 for the preparation of a rabies vaccine for immunization of horses.

19. The use of claim 18, wherein the recombinant horse serum albumin is used as a protective or stabilizing agent for rabies vaccine for immunization of horses.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to the field of recombinant expression protein, specifically to expression of a mammalian plasma protein, and in particular to recombinant horse serum albumin (ESA) and a preparation method thereof; in addition, the invention also relates to application of the recombinant horse serum albumin obtained by the method.

Background

Serum albumin is the most predominant protein in mammalian plasma and is widely used as an adjuvant or protective agent in the pharmaceutical industry to stabilize the active ingredients of drugs. Many viral vaccines employ albumin as a protective agent. One of them is the human rabies vaccine.

Rabies (Rabies) is a natural epidemic disease of the acute lethal Central Nervous System (CNS) in humans and all warm-blooded animals caused by the Rabies virus (RABV). The disease is distributed globally, and people are infected by being bitten by sick animals, and the disease can be transmitted by the same species and different species of animals, besides the bite, by inhalation, ingestion or vertical transmission. Human rabies patients die for almost 100%. China is one of the countries with serious prevalence of rabies, and the enhancement of the research and the prevention and the treatment of rabies is very urgent and has great burden. According to the WHO guidance principle for preventing and treating rabies, the exposing treatment is carried out immediately or as early as possible after the suspected sick animals grab and bite the animals. Except for the injection of rabies vaccine, the anti-rabies virus immunoglobulin should be locally infiltrated and injected immediately or as early as possible after wound treatment, so that the body is protected.

The anti-human rabies immune globulin is prepared by using human rabies vaccine to immunize horses and then collecting blood. Because human rabies vaccine is added with human serum albumin in the preparation process, the human rabies vaccine is used for immunizing horses, and the horses generate high-titer anti-human serum albumin antibodies, the anti-human serum finished products contain a certain amount of anti-human serum albumin antibodies, sensitization is easy to generate after injection, and the probability of anaphylactic reaction is very high. To overcome this problem, we developed genetic engineering expression studies of horse serum albumin. Recombinant horse serum albumin is used to replace human serum albumin to serve as a protective agent in the cell culture virus production process of rabies virus vaccines and in vaccine stock solution, so as to avoid the side effects.

In the invention patent CN201710147935, which is applied by the applicant on 3/13.2017, the applicant adopts a signal peptide of yeast α -mating factor (α -mating factor) as a signal peptide of Interferon, and the signal peptide is used for secretion expression of the Interferon to obtain a good effect, but in pichia pastoris secretion expression of horse serum albumin, the applicant uses a signal peptide of α -mating factor (α -mating factor) of yeast as a signal peptide of the albumin, and the expression level of target protein is very low.

The research aims to adopt pichia pastoris to express horse serum albumin in a recombination way, and the horse serum albumin is used for replacing human serum albumin to prepare the rabies virus vaccine for horses.

Disclosure of Invention

One of the technical problems to be solved by the present invention is to provide a method for preparing recombinant horse serum albumin (ESA).

The second technical problem to be solved by the invention is to provide the recombinant horse serum albumin prepared by the method.

The second technical problem to be solved by the invention is to provide an application of the recombinant horse serum albumin, and the recombinant horse serum albumin is used as a stabilizer or a protective agent for preparing rabies vaccines for immunizing horses. The recombinant horse serum albumin is used for replacing human serum albumin to be used as a protective agent in a cell culture virus production process of the rabies virus vaccine and a vaccine stock solution so as to avoid side effects of anaphylactic reaction.

In order to solve the technical problems, the invention adopts the following technical scheme:

in the first aspect of the invention, a preparation method of recombinant horse serum albumin is provided, wherein a nucleotide sequence which is optimized by codon and used for coding the horse serum albumin is constructed into an eukaryotic cell induced expression vector, then is transferred into the eukaryotic cell for culture, then is induced and expressed, and is purified, so that the recombinant horse serum albumin is obtained; the nucleotide sequence of the coded horse serum albumin which is optimized by the codon is a sequence shown as SEQ ID NO. 2.

As a preferred embodiment of the present invention, the culturing comprises a preliminary step of pre-inoculation.

As a preferred technical scheme of the invention, the eukaryotic cell is a yeast cell (such as saccharomyces cerevisiae, hansenula or pichia pastoris), preferably is a pichia pastoris cell, and most preferably is pichia pastoris GS 115.

In a preferred embodiment of the present invention, the expression mode may be intracellular expression or secretory expression, and the present invention is preferably a secretory expression method, wherein the eukaryotic cell inducible expression vector is a secretory expression vector, and after the inducible expression, the cells and particulate matter are removed by centrifugation, and the supernatant is collected and purified.

As a preferred technical scheme of the invention, the secretion expression vector is a pichia pastoris expression vector, the expression vector can be pHIL-D2, pPIC9, pGAP, pPIC3.5, preferably pHIL-D2 expression vector, the secretion expression vector contains a horse serum albumin expression cassette, and a signal peptide required by secretion expression can be yeast α -factor secretion signal peptide or a horse serum albumin self-secretion signal peptide, preferably a horse serum albumin self-secretion signal peptide.

As a preferred technical scheme of the invention, the codon-optimized nucleotide Sequence for coding horse serum albumin is optimized according to a Sequence (shown as a Sequence in SEQ ID NO: 1) provided by NCBI Reference Sequence NM-001082503.1 according to a codon preferred by yeast, EcoR I is added before an initiator and after a terminator, a cDNA expression cassette is artificially synthesized and inserted into an EcoR I site of a Pichia pastoris expression vector pHIL-D2 to construct an ESA horse serum albumin/pHIL-D2 expression vector; taking a certain amount of pHIL-D2 plasmid DNA, carrying out enzyme digestion linearization by Sal I, adding into pichia pastoris culture, transferring the plasmid DNA into yeast by an electric shock method, integrating the plasmid DNA into yeast genome DNA, and screening by using a histidine-deficient culture medium to obtain a cell strain for expressing albumin.

As a preferred embodiment of the present invention, the medium used for inducing expression after transferring into eukaryotic cells for culture may be BMGY/BMMY (a complex glycerol or methanol medium with buffering action consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% amino acid-free yeast nitrogen source, 4x 10-5% biotin, 1% glycerol or 0.5% methanol), BMG/BMM (a minimum glycerol or methanol medium with buffering action consisting of 100 mM potassium phosphate pH 6.0, 1.34% amino acid-free yeast nitrogen source, 4x 10-5% biotin, 1% glycerol or 0.5% methanol), or YPD/YPM (1% yeast extract, 2% peptone, 2% glucose or 0.5% methanol), the YPD/YPM medium system is preferably used in the present invention.

As a preferred technical scheme of the invention, the culture steps are as follows: selecting clone, inoculating into YPD culture medium, culturing at 30 deg.C and 200rpm for 24-72 hr, preferably 48 hr, centrifuging to remove supernatant, changing culture medium to YPM, culturing at 28 deg.C and 200rpm for 24-120 hr, preferably culturing at 28 deg.C and 200rpm for 48-96 hr, more preferably culturing at 28 deg.C and 200rpm for 72 hr.

As a preferred embodiment of the present invention, the inducible expression is achieved by gradually adding methanol during the yeast culture, preferably 0.5-1.5% methanol every 24 hours, more preferably 1% methanol every 24 hours.

As a preferred technical scheme of the invention, the purification method is to separate and purify culture supernatant obtained by a centrifugation method by anion-cation exchange chromatography. The method comprises capturing the target protein by cation exchange chromatography, wherein the cation exchange gel can be CM-Sepharose FF gel or SP-Sepharose FF gel, preferably SP-Sepharose FF gel. Anion exchange chromatography captures horse serum albumin. The anion exchange gel may be Q-Sepharose FF gel or DEAE-Sepharose FF gel, preferably DEAE-Sepharose FF gel.

In a second aspect of the present invention, there is provided a recombinant horse serum albumin produced by the above-described production method.

In a third aspect of the invention, the invention provides an application of the recombinant horse serum albumin in preparing rabies vaccines for immunizing horses. The recombinant horse serum albumin is used as a rabies vaccine protective agent or a stabilizing agent for producing horse anti-human rabies virus immunoglobulin for immunizing horses.

Compared with the prior art, the invention has the following beneficial effects: experiments prove that the horse anti-human rabies antiserum obtained by immunizing horses with the vaccine obtained by the method does not have an anti-human serum albumin antibody, and the quality meets the requirements of pharmacopoeia. The invention adopts the signal peptide of the target protein to express, and the expression level of the target protein is high. The recombinant horse serum albumin is used for replacing human serum albumin to serve as a protective agent in a cell culture virus production process of the rabies virus vaccine and a vaccine stock solution, so that a certain amount of anti-human serum albumin antibody is prevented from being contained in an anti-serum finished product, sensitization is easily generated after injection, and the side effect of very high allergic reaction probability is avoided. Adding 2% horse serum albumin (w/v) and 2% sorbitol (w/v) into rabies antigen, and preserving at 2-8 ℃ for 2 years with unchanged titer; after a horse is immunized by a rabies antigen with commercially available human serum albumin as a stabilizer, a horse anti-human serum albumin antibody with high titer is generated; leading the final product horse anti-human rabies serum to generate extremely high positive reaction when being applied in people. And the rabies antigen using horse serum albumin as a stabilizer is used for immunizing horses to obtain high anti-rabies virus antibodies, and the anti-human serum albumin antibody is not detected. The recombinant horse serum albumin obtained by the invention can replace human serum albumin to be used as a stabilizer of a rabies vaccine for immune horses, and is used for producing high-quality horse anti-human rabies virus serum.

Drawings

FIG. 1 is a schematic structural diagram of a recombinant Pichia pastoris expression vector according to example 2 of the invention.

FIG. 2 is an SDS-PAGE electrophoretogram for screening expression clones induced with methanol and detecting the amount of target protein expressed by the clones in example 3 of the present invention. In FIG. 2, from left to right, 1-9 wells show the results of 2. mu.l of culture supernatant obtained after 2 days of YPD culture and 3 days of YPM culture of clones 1-9; and the 10 th hole is a protein molecular weight standard. FIG. 2 shows clone 5 does not express the protein of interest; clone 1 expresses trace amount of target protein; clones 4 and 6 expressed higher amounts.

FIG. 3 is a SDS-PAGE image of clone 6 of example 4 of the present invention grown in YPD medium at high density for 2 days, and sampled by methanol induction. In FIG. 3, from left to right, the 1 st well is the protein molecular weight standard, and the 2 nd to 9 th wells are clones 1 to 8, respectively. It can be seen from the figure that the expression levels of the target proteins of 8 clones were substantially the same.

FIG. 4 is SDS-PAGE of a purified target protein obtained by DEAE-Sepharose FF ion exchange chromatography in example 5 of the present invention; in FIG. 4, the 1 st well is DEAE-Sepharose purified recombinant horse serum albumin; and the 2 nd hole is a protein molecular weight standard.

FIG. 5 is a SEC-HPLC (high pressure liquid phase) chromatogram of recombinant horse serum albumin in example 5 of the present invention. 20 microgram of recombinant horse serum albumin was chromatographed on Agilent1260 high performance liquid chromatography using TSK gel UP-SW3000 (4.6 mm I.D.. times.15 cm) chromatography column. The target protein peaks at 11.215 minutes and has a purity of more than 99%.

Detailed Description

In order to clearly understand the technical contents of the present invention, the preparation of the recombinant equine serum albumin, the preparation of equine rabies vaccine and the immunization method are specifically taken as examples, and the following specific examples are given for detailed description. However, the specific embodiments are merely illustrative, and not restrictive of the invention.