CN117467018A

CN117467018A - Recombinant subunit fusion protein rEG-Fc and preparation method thereof

Info

Publication number: CN117467018A
Application number: CN202210909721.9A
Authority: CN
Inventors: 殷波; 翟伟锋; 王�锋; 张震; 仲从浩
Original assignee: Shanghai Shen Lian Biomedical Corp
Current assignee: Shanghai Shen Lian Biomedical Corp
Priority date: 2022-07-29
Filing date: 2022-07-29
Publication date: 2024-01-30

Abstract

The invention discloses a recombinant subunit fusion protein rEG-Fc and a preparation method thereof; the fusion protein rEG-Fc contains echinococcus granulosus EG95 antigen and sheep antibody Fc protein fragments, and the amino acid sequence of the fusion protein rEG-Fc protein fragments is shown in SEQ ID NO. 1. The invention obtains a monoclonal cell strain capable of secreting and expressing subunit fusion protein rEG-Fc, has high expression yield, and can obtain a large amount of fusion proteins through one-step affinity chromatography.

Description

Recombinant subunit fusion protein rEG-Fc and preparation method thereof

Technical Field

The invention belongs to the technical field of biological products for animals, relates to recombinant subunit fusion protein rEG-Fc and a preparation method thereof, and particularly relates to recombinant echinococcus granulosus EG95-Fc fusion protein and a preparation method thereof.

Background

Echinococcosis (Echinococcosis) is a serious parasitic disease of human and animals caused by the parasitic of Echinococcosis (Echinococcosis) larvae of Echinococcosis in tissues and organs such as lung and liver of human and animals. Echinococcosis is widely spread and distributed worldwide, and the world animal health organization (World Organization for Animal Health; french: office international des e shoes, OIE) classifies echinococcosis as a global reported infectious disease and as a plurality of zoonosis; the world health organization (World Health Organization, WHO) ranks echinococcosis as one of the diseases that the global early warning system prioritizes and treats with emergency. Echinococcosis is also one of five major parasitic diseases planned to be prevented and treated by the ministry of health of China.

Echinococcosis is mainly divided into echinococcosis granulosa (cystic echinococcosis, CE) and echinococcosis multiflorum according to the focus morphology and infection pathogen difference, wherein the echinococcosis granulosa is most widely distributed and the number of patients is the greatest. The causative agent of CE is currently composed of several echinococcus complex species: echinococcus granulosus (Echinococcus granuLosus), echinococcus Canadian (Canadensis), echinococcus Ma Jiqiu, echinococcus octreodorus, wherein CE caused by Echinococcus granulosus type G1 exceeds 90% and CE caused by Echinococcus Canadian type G6 exceeds 7%.

The current research shows that the epidemic of echinococcosis is controlled mainly by cutting off the development link of echinococcosis, controlling the infection of intermediate hosts such as human beings, livestock and the like on the echinococcosis, preventing or expelling parasites to treat final hosts such as dogs and the like, and blocking the wide-range spreading of ova. Wherein the epidemic of echinococcosis granulosa can be effectively controlled for the vaccination of intermediate hosts. Lighters et al found EG95 to be one of the natural hexacercaria antigens present in EG and the most potent protective antigen among the many proteins screened, and have successfully developed vaccines against echinococcosis in sheep. However, the existing recombinant EG95 protein vaccine is prepared by renaturation of an escherichia coli inclusion body, has low purity, is difficult to maintain the spatial structure of the protein, and has a certain limitation because the immunogenicity duration of the antigen is short.

Disclosure of Invention

In order to solve the problems of low antigen expression level, poor solubility and short immune duration in echinococcus granulosus subunit vaccines in the prior art, the invention provides subunit fusion protein rEG-Fc capable of stably and efficiently expressing echinococcus granulosus EG95 antigen and sheep immunoglobulin Fc and a construction and expression method thereof in a CHO or 293T cell system. The invention screens mutants capable of obviously increasing the immune duration of Fc fusion protein aiming at Fc fragments in sheep IgG, and the sequences of the mutants are shown as SEQ ID NO. 4. The monoclonal cell strain capable of secreting and expressing subunit fusion protein rEG-Fc has high expression yield, and a large amount of fusion proteins can be obtained through one-step affinity chromatography.

The aim of the invention is realized by the following technical scheme:

< first aspect >

The invention provides a recombinant subunit fusion protein rEG-Fc, which contains echinococcus granulosus EG95 antigen and sheep antibody Fc protein fragment, wherein the antibody Fc protein fragment

The heavy chain constant region fragment of sheep IgG has an amino acid sequence shown in SEQ ID NO. 3;

or, the amino acid sequence of the mutant of the heavy chain constant region fragment of sheep IgG is shown as SEQ ID NO. 4.

As one embodiment, the amino acid sequence of the echinococcus granulosus EG95 antigen is shown in SEQ ID NO. 2.

As one embodiment, the EG95 antigen is linked to an antibody Fc protein fragment of sheep via a linker rich in glycine and serine.

As one embodiment, the linker sequence is GGGSGGGSGGGS.

As one embodiment, the EG95 antigen is located at the N-terminus of the linker, and the Fc protein fragment is located at the C-terminus of the linker; or the EG95 antigen is positioned at the C end of the linker, and the Fc protein fragment is positioned at the N end of the linker.

As one embodiment, the fusion protein rEG-Fc,

is a protein with an amino acid sequence shown as SEQ ID NO. 1;

or, the amino acid sequence shown as SEQ ID NO.1 comprises a derivative protein which is subjected to substitution, deletion or addition of one amino acid or a plurality of amino acids and has immunogenicity;

or, a derivative protein in which EG95 and Fc are reversed in order on the amino acid sequence shown in SEQ ID NO.1 and Fc is placed at the amino terminus.

As one embodiment, the fusion protein rEG-Fc is a protein having the amino acid sequence shown in SEQ ID NO. 12.

As one embodiment, the fusion protein rEG-Fc has one or more tags attached to the amino-or carboxy-terminus of the amino acid sequence.

As one embodiment, the tag is selected from the group consisting of poly-Arg, poly-His, flag, c-myc and HA.

According to the technical scheme of the recombinant subunit fusion protein rEG-Fc of the present invention, one or more tag amino acids of poly-Arg, poly-His, flag, c-myc and HA are preferably linked at the amino-terminus or the carboxy-terminus of the amino acid sequence shown in SEQ ID NO. 1.

As one embodiment, the expression system of the fusion protein rEG-Fc includes, but is not limited to, mammalian cells and insect cells.

As one embodiment, the insect cells are Sf9 cells, sf21 cells, high Five cells.

As one embodiment, the mammalian cells are CHO cells, 293T cells. More preferably, the mammalian cells are CHO cells.

< second aspect >

The invention provides a preparation method of recombinant subunit fusion protein rEG-Fc, which comprises the following steps:

s1, cloning a gene coded by rEG-Fc fusion protein into a eukaryotic expression vector to obtain a recombinant plasmid containing rEG-Fc fusion protein coding gene;

s2, transfecting a recombinant plasmid containing a rEG-Fc fusion protein coding gene into a CHO cell strain;

s3, obtaining a highly expressed cell strain by culturing, screening and domesticating the CHO cell strain obtained in the step S2;

s4, fermenting and culturing the cell strain with high expression, and purifying to obtain rEG-Fc fusion protein.

As one embodiment, in step S1, the rEG-Fc fusion protein encodes a gene as shown in SEQ ID NO.5 or SEQ ID NO. 6.

As one embodiment, the eukaryotic expression vector is a mammalian cell expression vector. Preferably, the eukaryotic expression vector is pee6.4, pee12.4, pgl4.13, pcdna3.1, more preferably, the eukaryotic expression vector is pcdna3.1.

As one embodiment, the cell used to express the recombinant subunit fusion protein rEG-Fc is a CHO cell, preferably the CHO cell line is a DG44, DXB11, CHO-K1 or CHO-S cell line. More preferably, the CHO cell line is CHO-K1.

< third aspect >

The invention provides a CHO cell strain 3F 4-rEG-Fc (CHO 3F 4-rEG-Fc) with a preservation number of CCTCC NO: C2022186.

In the invention, chinese hamster ovary cells 2H 1-rEG are submitted to China center for preservation, the preservation address is China university of Wuhan, the preservation number is CCTCC NO: C2022185, and the preservation date is 2022, 6 months and 15 days.

Chinese hamster ovary cells 3F 4-rEG-Fc have been submitted to China center for type culture Collection with a preservation address of university of Wuhan, china, a preservation number of CCTCC NO: C2022186, and a preservation date of 2022, 6 months and 15 days.

Compared with the prior art, the invention constructs and screens a CHO cell strain which stably and efficiently secretes and expresses subunit fusion protein rEG-Fc of echinococcus granulosus EG95 in a suspension manner, the cell strain expresses recombinant subunit fusion protein rEG-Fc with high yield (the yield is up to 0.5-1 g/L), the purification is easy (the purity of target protein in cell culture supernatant can reach more than 80 percent, the purity of the target protein can reach more than 90 percent only by one-step affinity chromatography, the requirements of subunit vaccines and diagnostic reagents are far met), and the large-scale production is easy; the invention constructs rEG-Fc fusion protein mutant, and compared with wild fusion protein, the mutant has obviously improved immune duration. In addition, the CHO cell strain for production has high controllability, easy quality control, stable production protein batch to batch and high biological safety (no virus and no risk of virus dispersion) during culture.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is a schematic diagram of pcDNA3.1-rEG 95.95-Fc plasmid map;

FIG. 2 shows agarose gel electrophoresis of a double enzyme digestion assay of pcDNA3.1-rEG 95-Fc-M; lane M: a DNA Marker; lane 1: pcDNA3.1-rEG-Fc-M double digested strips.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several modifications and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention. The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

The invention adopts the following technical scheme:

example 1, optimization and Synthesis of Gene sequences.

The invention optimizes the codon of the base sequence of the coding recombinant subunit fusion protein rEG-Fc by taking CHO as a host cell, the sequence is shown as SEQ ID NO.5, and the gene is rEG-95-Fc-WT in the example; the nucleotide sequence of the recombinant subunit fusion protein rEG-Fc mutant is subjected to codon optimization, the sequence is shown as SEQ ID NO.6, and the gene is rEG-95-Fc-M in the example; the nucleotide sequence of the coded recombinant subunit protein rEG is optimized by codons, and the sequence is shown as SEQ ID NO. 7. The optimized base sequence is synthesized by Nanjing Jinsri biotechnology limited company.

Example 2 construction of recombinant expression vector.

2.1 construction of recombinant expression plasmid pcDNA3.1-rEG95-Fc-WT and pcDNA3.1-rEG-Fc-M.

(1) Respectively taking rEG-Fc-WT and rEG-Fc-M as templates, designing an upstream primer rEG-Fc-Nhe I-F and a downstream primer rEG-Fc-Xho I-R, and carrying out PCR amplification to obtain gene fragments rEG-Fc-WT and rEG-Fc-M, wherein a restriction enzyme Nhe I site and a protective base are introduced into the 5' end of the upstream primer, and the Nhe I site sequence is GCTAGC; the 5' end of the downstream primer is introduced with a restriction enzyme Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Table 1 and Table 2, wherein single underlined is the protective base sequence, italics is the cleavage site sequence, and bold is the stop codon.

Table 1: PCR primer name and sequence

Table 2: PCR reaction procedure

(2) The amplified gene fragments rEG-Fc-WT and rEG-Fc-M were digested with the endonucleases Nhe I and the endonucleases Xho I, the digested gene fragments were recovered, ligated to pcDNA3.1 vector treated with the same endonucleases Nhe I and endonucleases Xho I, E.coli DH 5. Alpha. Competent cells were transformed with the ligation product, plated on LB medium plates containing 100. Mu.g/ml ampicillin, cultured at 37℃and single colonies were picked up in 3ml liquid LB medium containing 100. Mu.g/ml ampicillin when colonies were clearly visible on the plates, cultured at 37℃and plasmids were subsequently extracted. The recombinant plasmids pcDNA3.1-rEG95-Fc-WT and pcDNA3.1-rEG-Fc-M are obtained, and the recombinant plasmids are confirmed to be consistent with the target sequence through sequencing verification. The plasmid map is shown in FIG. 1, and the double cleavage assay is shown in FIG. 2.

2.2 construction of recombinant expression plasmid pcDNA3.1-rEG95.

(1) Designing an upstream primer rEG-Nhe I-F and a downstream primer rEG-Xho I-R by taking rEG95 as templates, and carrying out PCR amplification to obtain a gene fragment rEG95, wherein a restriction endonuclease Nhe I site and a protective base are introduced into the 5' end of the upstream primer, and the sequence of the Nhe I site is GCTAGC; the 5' end of the downstream primer is introduced with a restriction enzyme Xho I site, a stop codon and a protective base, wherein the Xho I site sequence is CTCGAG. The primer sequences and PCR reaction procedures are shown in Table 3 and Table 4, wherein single underlined is the protective base sequence, italics is the cleavage site sequence, and bold is the stop codon.

Table 3: PCR primer name and sequence

Table 4: PCR reaction procedure

(2) The amplified gene fragment rEG was digested with the endonucleases Nhe I and the endonucleases Xho I, the digested gene fragment was recovered, ligated to pcDNA3.1 vector treated with the same endonucleases Nhe I and endonucleases Xho I, E.coli DH 5. Alpha. Competent cells were transformed with the ligation product, plated on LB medium plates containing 100. Mu.g/ml ampicillin, cultured at 37℃until colonies on the plates were clearly visible, and single colonies were picked up on 3ml liquid LB medium containing 100. Mu.g/ml ampicillin, cultured at 37℃and plasmids were subsequently extracted. The recombinant plasmid pcDNA3.1-rEG is obtained, and the recombinant plasmid is confirmed to be consistent with a target sequence through sequencing verification.

2.3 preparation of plasmid for transfection.

(1) 2ml of plasmid pcDNA3.1-rEG95-Fc-WT, 2ml of plasmid pcDNA3.1-rEG-Fc-M and 2ml of plasmid pcDNA3.1-rEG95 were extracted, respectively, with reference to the kit instructions for endotoxin-free plasmid.

(2) 1. Mu.l of the sample was subjected to agarose gel electrophoresis.

(3) The concentration of the extracted plasmid was determined using nanodrop, wherein the concentration of the plasmid pcDNA3.1-rEG95-Fc-WT was 801ng/ul, the concentration of pcDNA3.1-rEG-Fc-M was 781ng/ul, and the concentration of the plasmid pcDNA3.1-rEG95 was 654ng/ul.

Example 3: establishment of recombinant plasmid transfection CHO-K1 cells and monoclonal screening.

3.1CHO-K1 cell transfection

(1) The cells used for transfection are CHO-K1 cells which have been domesticated to be capable of suspension culture.

(2) Day before transfection (Day-1), CHO-K1 cells were plated at 1X 10 ⁶ Each viable cell/ml density was inoculated into shake flasks containing 30ml basal medium.

(3) On the Day of transfection (Day 0), the viable cell density and the viable rate were examined, and the viable cell density was 2X 10 ⁶ -6×10 ⁶ The cell viability should reach 95-99% per viable cell/ml.

(4) Diluting 30 mug of recombinant plasmid by using 1500 mug of basic culture medium, and fully and uniformly mixing to obtain a diluent; 60 mu L of PEI transfection reagent (1 mg/mL) is immediately added into the diluent, vortexed for 10 seconds and fully mixed; incubation for 15min at room temperature allowed the formation of DNA-PEI cationic nucleic acid transfection reagent complexes.

(5) The complex was added to the cell fluid and cultured with shaking.

(6) Day 2 (Day 2) after transfection, samples were taken and subjected to SDS-PAGE electrophoresis.

3.2 cell line selection

(1) On Day 2 post-transfection (Day 2), G418 solution was added at a final concentration of 500. Mu.g/ml.

(2) On Day 7 after transfection (Day 7), viable cell density and viability were examined according to 1X 10 ⁶ The individual viable cells/ml density were inoculated into shake flasks of basal medium.

(3) Passaging every 3-4 days until the cell viability is recovered to more than 90%, and the viable cell density reaches 1×10 ⁶ Each living cell/ml.

(4) Screening the monoclonal cell strain by adopting a limiting dilution method to obtain subclones with the highest recombinant protein expression level in the supernatant.

(5) The cell strains with high expression rEG-Fc-WT are obtained by screening, and the total three strains are respectively: 1F7,5D1,7A10, screening to obtain three cell strains with high expression of rEG-Fc-M: 2B11,3E7,3F4, screening to obtain three cell strains with high expression of rEG 95: 2H1,3B12,4C6 strain. The 2H1 cell strain is submitted to China Center for Type Culture Collection (CCTCC) with a collection address of university of Wuhan, china and a collection number of CCTCC NO: C2022185.

(6) Freezing and preserving the cell strain obtained by screening under the condition of liquid nitrogen.

(7) The 3F4 cell strain is submitted to China Center for Type Culture Collection (CCTCC) with a collection address of university of Wuhan, china and a collection number of CCTCC NO: C2022186.

Example 4 cell shake flask fermentation

(1) Cells of lines 1F7,5D1,7A10, 2B11,3E7,3F4, 2H1,3B12, and 4C6 were prepared at a ratio of 0.5X10 ⁶ Each viable cell/mL cell density was inoculated into 125mL shake flasks containing 30mL of medium.

(2) Shake flask placed at 37 ℃,8% CO ₂ Shake culture in a shaker at 120 rpm.

(3) The cell viability was measured daily until the cell viability was less than 80%, and the culture supernatant was harvested by centrifugation and subjected to the next purification.

Example 5 protein purification

(1) The fermentation broth was harvested, centrifuged at 8000rpm for 30min and filtered through a 0.2 μm filter.

(2) And (5) loading. Purification was performed using a 5mL pre-packed nickel column, flow rate.

(3) And (5) washing impurities. Buffer A (500mM NaCl,25mM PB (pH 7.2), 50mM imidazole), 25ml in volume, flow rate 2ml/min.

(4) Eluting. Buffer B (500mM NaCl,25mM PB (pH 7.2), 500mM imidazole), 50ml in volume, 2ml/min flow rate. The elution peaks were collected.

(5) Balance. Buffer C (500mM NaCl,25mM PB (pH 7.2)) was 50ml in volume and 2ml/min in flow rate.

(6) And (5) preserving. 20% ethanol, volume 10ml, flow rate 2ml/min.

(7) SDS-PAGE electrophoresis is performed on the collected peak samples, and the concentration and purity of the recombinant protein are calculated according to a gray-scale analysis method.

(8) And (5) purifying the result. The purified protein content of the recombinant protein expressed per ml of culture volume was calculated from the purified collection volume, the protein concentration and the culture volume, as shown in Table 5.

TABLE 5 results after purification of recombinant proteins

Cell strain	1F7	5D1	7A10	2B11	3E7	3F4	2H1	3B12	4C6
										Protein content (mg/ml)	0.58	0.52	0.49	0.70	0.62	0.75	0.33	0.19	0.25
Purity of purified protein (%)	90	95	91	94	94	95	91	92	94

Example 6 immunogenicity analysis of recombinant proteins.

6.1 preparation of immune samples. Diluting the purified recombinant protein to 100 mug/ml by PBS, and emulsifying with sterilized Montanide ISA 50V adjuvant according to the volume ratio of 1:1 to obtain the immune required sample.

6.2 sheep immunization experiments. Four groups of 5 immunized lambs were assigned to each of the prepared samples rEG-Fc-WT, rEG95-Fc-M, rEG and PBS. The immunization and blood sampling flow comprises: 1) Taking about 5ml of blood before immunization; 2)

Day 1 first immunization: immunization of 1ml per lamb; 3) Boosting on day 28: immunization of 1ml per lamb; 4) 10ml of blood was collected per lamb on day 42, and antibody titers were detected by ELISA; 4) On day 168, 10ml of blood was collected per lamb and the antibody titers were measured by ELISA.

6.3 antibody titer detection. The ELISA method is used for detecting the antibody titer of immune serum, and the results are shown in table 6, which shows that the recombinant protein has better immunogenicity, the antibody titer generated by rEG-Fc-WT and rEG-Fc-M proteins is higher than that generated by rEG-95 proteins, and the form of Fc fusion protein can improve the immunogenicity of the protein; whereas the rEG95-Fc-M protein had an antibody titer higher than rEG-Fc-WT protein after 168 days, indicating that the mutants of the selected Fc fusion proteins of the present invention can increase the immune duration of the protein.

Table 6: antibody titer detection results.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. A recombinant subunit fusion protein rEG-Fc comprising an echinococcus granulosus EG95 antigen and an antibody Fc protein fragment of sheep; the antibody Fc protein fragment

2. The recombinant subunit fusion protein rEG-Fc according to claim 1, wherein said echinococcus granulosus EG95 antigen has the amino acid sequence shown in SEQ ID No. 2.

3. The recombinant subunit fusion protein rEG-Fc according to claim 1, wherein said EG95 antigen is linked to an antibody Fc protein fragment of sheep via a linker enriched in glycine and serine; the linker sequence is GGGSGGGSGGGS.

4. The recombinant subunit fusion protein rEG-Fc according to claim 3, wherein said EG95 antigen is at the N-terminus of a linker and said Fc protein fragment is at the C-terminus of a linker; or the EG95 antigen is positioned at the C end of the linker, and the Fc protein fragment is positioned at the N end of the linker.

5. The recombinant subunit fusion protein rEG-Fc of claim 1, wherein the fusion protein rEG-Fc,

is a protein with an amino acid sequence shown as SEQ ID NO. 1;

6. The recombinant subunit fusion protein rEG-Fc according to any one of claims 1-5, wherein said fusion protein rEG-Fc has one or more tags attached to the amino-or carboxy-terminus of the amino acid sequence; the tag is selected from the group consisting of poly-Arg, poly-His, flag, c-myc and HA.

7. The recombinant subunit fusion protein rEG-95-Fc of claim 1, wherein the expression system of fusion protein rEG-Fc includes, but is not limited to, mammalian cells and insect cells; the insect cells are Sf9 cells, sf21 cells or High Five cells; the mammalian cells are CHO cells or 293T cells.

8. A method of preparing a recombinant subunit fusion protein rEG-Fc according to any one of claims 1-7, comprising the steps of:

s1, cloning a coding gene of rEG-Fc fusion protein into a eukaryotic expression vector to obtain a recombinant plasmid containing rEG-Fc fusion protein coding gene;

9. The method of claim 8, wherein the eukaryotic expression vector is pee6.4, pee12.4, pgl4.13, or pcdna3.1; the CHO cell strain is DG44, DXB11, CHO-K1 or CHO-S cell strain.

10. Chinese hamster ovary cell 3F4-rEG95-Fc has a preservation number of CCTCC NO: C2022186.