CN115851831A - Construction method and application of CHO cell strain for efficiently expressing foreign protein - Google Patents

Construction method and application of CHO cell strain for efficiently expressing foreign protein Download PDF

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Publication number
CN115851831A
CN115851831A CN202210982902.4A CN202210982902A CN115851831A CN 115851831 A CN115851831 A CN 115851831A CN 202210982902 A CN202210982902 A CN 202210982902A CN 115851831 A CN115851831 A CN 115851831A
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protein
seq
virus
vaccine composition
cho cell
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田克恭
谭菲菲
张许科
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Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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Abstract

The invention relates to a construction method and application of a CHO cell for efficiently expressing foreign protein, wherein the method comprises the following steps: adding a signal peptide sequence at the N end of an exogenous protein sequence; step (2) fusion-connecting the foreign protein added with the signal peptide sequence and Fc to construct an expression plasmid; step (3) transfecting CHO cells with the expression plasmid constructed in the step (2); and (4) harvesting the foreign protein. The present invention also relates to a method for expressing a foreign protein using the constructed CHO cell, and a vaccine composition comprising the foreign protein.

Description

Construction method and application of CHO cell strain for efficiently expressing foreign protein
The invention relates to Chinese patent application No. 202010055178.1, which is a divisional application of Chinese patent application entitled "construction method of CHO cell strain capable of efficiently expressing foreign protein and application thereof", and its application in 2020 and 17.01.1.2020.
Technical Field
The invention belongs to the field of cell lines and preparation methods thereof, and particularly relates to a cell modified by introducing foreign genetic materials, and a construction method and application thereof.
Background
CHO cells are isolated from the ovaries of adult female hamsters and are epithelial adherent cells. The cell is immortal, can be passaged for more than one hundred generations, and is a cell widely used in bioengineering at present. Compared with other expression systems, the expression system has the following advantages: (1) Has accurate post-transcriptional modification function, and the expressed protein is closest to the natural protein of eukaryote in the aspects of molecular structure, physicochemical property and biological function; (2) The culture medium can not only grow in a wall-attached manner, but also be cultured in a suspension manner, and can bear higher shearing force and osmotic pressure; (3) has the high-efficiency amplification and expression capacity of the recombinant gene; (4) Has the extracellular secretion function of the product, rarely secretes the endogenous protein of the product, and is convenient for separating and purifying downstream products.
However, how to improve the expression level of the foreign protein expressed by the CHO expression system is always the direction of research by scholars at home and abroad, most of the methods aim at improving the expression level of the foreign protein by specifically modifying specific foreign genes, are not suitable for high-efficiency expression of other foreign proteins, and when the CHO expression system is applied to other foreign proteins, the genes of other foreign proteins need to be modified again, so that the process is complicated. Therefore, a high-efficiency expression method which is suitable for different foreign proteins and is simple to operate is needed in practical production.
Disclosure of Invention
In order to solve the above problems, the present invention provides a CHO cell construction method for efficiently expressing a foreign protein, wherein the method comprises: adding a signal peptide sequence at the N end of an exogenous protein sequence; step (2), fusing and connecting the foreign protein sequence added with the signal peptide sequence and the Fc sequence together to construct an expression plasmid; step (3) transfecting CHO cells with the expression plasmid constructed in the step (2); and (4) harvesting the foreign protein.
In the step (4), screening clone strains with higher yield; and (3) evaluating the stability and the yield of the obtained clone strains so as to select the clone strains with better stability, good cell growth characteristics and higher protein yield for production.
The method of the invention can realize high-efficiency expression of the foreign protein, can obviously improve the expression quantity of the foreign protein compared with the conventional CHO expression, can be applied to high-efficiency expression of various foreign proteins, can be widely applied to various veterinary vaccine proteins, does not need to optimize the gene of the foreign protein, and has wide application range.
As an embodiment of the invention, in the CHO cell construction method of the invention, the signal peptide sequence in the step (1) is shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No. 14. The signal peptide shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No.14 can be optionally applied to a CHO cell construction method for efficiently expressing a specific foreign protein, and is not limited to a specific signal peptide sequence used in each example.
As an embodiment of the invention, in the CHO cell construction method, the Fc sequence in the step (2) is shown in SEQ ID No.15 or SEQ ID No. 16. The Fc sequence shown in SEQ ID No.15 and the Fc sequence shown in SEQ ID No.16 can be optionally applied to a CHO cell construction method for efficiently expressing a specific foreign protein, and are not limited to the specific Fc sequences used in the respective examples.
As an embodiment of the present invention, in the CHO cell construction method of the present invention, the foreign protein gene in step (1) includes african swine fever virus CD2v protein, avian adenovirus Penton protein, avian adenovirus Fiber-2 protein, avian egg-loss syndrome virus Penton protein, avian egg-loss syndrome virus tFiber protein, chicken infectious bursal disease virus VP2 protein, porcine circovirus type 3 Cap protein, porcine circovirus type 2 Cap protein, porcine pseudorabies virus gB protein, porcine pseudorabies virus gD protein, porcine parvovirus VP2 protein, porcine pestivirus E2 protein, bovine infectious rhinotracheitis virus gB protein, bovine infectious rhinotracheitis virus gD protein, foot and mouth disease virus VP0 protein, foot and mouth disease virus VP3 protein, foot and mouth disease virus VP1 protein, rabbit plague virus VP60 protein, japanese blood fluke GALE protein, japanese schistosoma Wnt5 protein.
The invention also relates to the CHO cell which is prepared/constructed by the method and can efficiently express the foreign protein.
The invention also relates to a method for efficiently expressing the foreign protein, wherein the method uses the CHO cell to express the foreign protein.
The invention also relates to application of the constructed CHO cell in preparation of foreign protein.
The invention also relates to application of the method for efficiently expressing the foreign protein in preparation of the foreign protein.
The invention also relates to a vaccine composition, which comprises the foreign protein prepared by the method and a pharmaceutically acceptable carrier. In particular, the vaccine composition is a subunit vaccine composition.
The foreign protein prepared by the preparation method has the advantages of biological safety, immunogenicity, immune efficacy and no adverse reaction on the growth and development of animals, and can be used for preparing subunit vaccines.
As one embodiment of the present invention, in the vaccine composition of the present invention, the carrier includes an adjuvant including: (1) Mineral oil, alumina gel adjuvant, saponin, alfudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer (Block co-polymer), SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, montanide ISA 206 and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100.
The adjuvant content is 5% -70% V/V.
The adjuvant content can be selected from 5% V/V, 6% V/V, 7% V/V, 8% V/V, 9% V/V, 10% V/V, 15% V/V, 20% V/V, 25% V/V, 30% V/V, 35% V/V, 40% V/V, 45% V/V, 50% V/V, 55% V/V, 60% V/V, 65% V/V, 66% V/V, 67% V/V, 70% V/V.
The invention also relates to a vaccine composition prepared from the prepared foreign protein, and a subunit vaccine composition is prepared by adding a pharmaceutically acceptable carrier into the foreign protein prepared by the method.
The vaccine compositions of the present invention may be formulated using available techniques, preferably together with a pharmaceutically acceptable carrier. For example, the oil may help stabilize the formulation and additionally serve as a vaccine adjuvant. The oil adjuvant can be natural or artificially synthesized. The term "adjuvant" refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) aluminium hydroxide, saponin (Saponine) (e.g. QuilA), alfvudine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivatives, (3) vaccines can be made in the form of oil-in-water, water-in-oil or water-in-oil-in-water emulsions, or (4) montanide tmgel.
In particular, the emulsion may be based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular esters of polyoxyethylated fatty acids (e.g. oleic acid), sorbitan, mannitol (e.g. anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic R, in particular L121 (cf. Hunter et al, 1995, "The therapy and Practical Application of additives" (Steved-Tull, eds. D.E.S. eds.) John Wiley and sons, NY,51-94 Todd et al, vaccine,1997, 15, 564-570).
In particular, the acrylic or methacrylic acid polymers are crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.
The amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount. By "effective amount" is meant the amount of adjuvant necessary or sufficient to exert their immunological effect in a host when administered in combination with the antigen of the invention without causing undue side effects. The precise amount of adjuvant to be administered will vary depending on factors such as the ingredients used and the type of disease being treated, the type and age of the animal to be treated, the mode of administration, and the other ingredients in the composition.
The subunit vaccine compositions of the invention may further comprise other agents added to the compositions of the invention. For example, the compositions of the present invention may also comprise agents such as: drugs, immunostimulants (e.g., alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin 2 (IL 2)), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. To prepare such compositions, methods well known in the art may be used.
The vaccine composition, in particular the subunit vaccine composition, according to the invention may be in an oral or non-oral dosage form.
Preferred are non-oral dosage forms that can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or epidural routes.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.
In order that the invention may be more readily understood, reference is now made to the following examples which are intended to illustrate the invention. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 construction method of CHO cell line efficiently expressing CD2v protein of African swine fever virus
Synthesis of CD2v protein Gene sequences
The sequence synthesis of the CD2v protein gene shown in SEQ ID No.17 is carried out, and the signal peptide sequence shown in SEQ ID No.1 is added at the N end of the CD2v sequence.
Construction of expression vector for CD2v fusion Fc
Design of primer CD2v to which a signal peptide sequence shown in SEQ ID No.1 was added and Fc gene shown in SEQ ID No.15 were ligated by fusion, the CD2v gene was first amplified by primer SF1 (5 'GCTCTAGAGCACCATGGACTGGACCTGGAGGATCC3')/SR 1 (5 'GTAGGTCCTTGGGCTCGTACAGTTGAAGAAAG3'), fc was amplified by primer SF2 (5 'CTTTCTTCAAACTGTACGAGCAAGGGGACCTACCAGGGACCTACC 3')/SR 2 (5 'GCAAGCTTACTTGCCGGGTCCTGAGAAGG3'), and the CD2v-mFc fusion product was amplified by primer SF1/SR2 using the two previous rounds of PCR products as templates. The amplified product is cut by EcoRI and Xho I and then connected with a pCAGGS vector, the connection product is identified by sequencing, and the plasmid with the correct sequence is named as pCAGGS-CD2v-Fc.
3. Transfection of CHO-S cells for Stable protein expression
After the correctly sequenced plasmid pCAGGS-CD2v-Fc is transformed, the plasmid is extracted by an endotoxin-free plasmid extraction kit, and the concentration of the plasmid is determined to be at least 1 mu g/mu l. The extracted plasmid was linearized using an NruI endonuclease.
The cell viability rate during transfection is higher than 95%, according to 1X 10 6 The density of individual viable cells/ml was inoculated into 125ml culture flasks and supplemented with CD FortiCHO complete medium to 30ml. Add 50. Mu.g of linearized plasmid to OptiPRO SFM medium to a final volume of 1.5ml and mix gently. Add 50. Mu.l FreeStyle MAX transfection reagent to 1.45ml OptiPRO SFM to a final volume of 1.5ml and mix gently. Immediately add the diluted FreeStyle MAX reagent solution to the plasmid DNA dilution solution, mix well, incubate the mixture at room temperature for 10 minutes to form DNA-lipid complexes, add 3ml OptiPRO SFM medium containing transfection reagent drop by drop to 125ml culture flasks containing cells. Placing the transfected cell culture at 37 ℃ with 8% CO 2 Was incubated on a shaking shaker at 130rpm. After 48 hours, drug screening was initiated.
The cells were centrifuged and resuspended, two T-150 flasks were inoculated at a density of 5X 10 5 Viable cells/ml. In the 1 st T-150 flask, puromycin was added to a final concentration of 10. Mu.g/ml, and MTX (methotrexate) was added to 100nmol/L. In the 2 nd T-150 flask, puromycin was added to a final concentration of 20. Mu.g/ml and MTX to 200nmol/L was added. Then subjecting the T-flask to 37 ℃ and 5% CO 2 And (4) incubating in a static incubator. When the cells showed signs of resuscitation, the cells were transferred to 125ml shake flasks at a seed density of 3X 10 5 Viable cells/ml. Placing the cells at 37 ℃ with 8% CO 2 Was incubated on a shaking shaker at 130rpm. Cells in flasks were passaged every 3-4 days at a seeding density of 3X 10 for each passage 5 Viable cells/ml. When the cell survival rate exceeds 85%, the living cell density exceeds 1 × 10 6 At individual viable cells/ml, drug screening stage 1 is complete.
Screening stage1 each cell pool was inoculated with 2 new 125ml shake flasks at a density of 4X 10 5 Viable cells/ml. In the 1 st flask puromycin was added to a final concentration of 30. Mu.g/ml and MTX to 500nmol/L was added. In the 2 nd flask puromycin was added to a final concentration of 50. Mu.g/ml and MTX to 1000nmol/L was added. Placing the cells at 37 ℃ with 8% CO 2 Was incubated on a shaking shaker at 130rpm. Sampling and counting every 3-4 days, and inoculating the cells in the subculture bottle every 3-4 days at the inoculation density of 3 × 10 when the cells show resuscitation signs 5 Viable cells/ml. When the cell viability reached 90%, the stage 2 screening was completed.
Recovering the cells of each cell pool obtained in the 2 nd stage, after the cell viability rate is more than 90%, according to the 3X 10 5 Viable cells/ml density were inoculated into a 125ml shake flask containing 30ml fresh medium, the cells were placed at 37 ℃ with 8% CO 2 The incubation was performed on a shaking shaker at 130rpm. Samples were taken periodically (days 0, 3, 5, 7, 10, 12, 14) until the culture viability dropped below 50%. After sampling, glucose was added to the culture: on days 3, 5 and 7, 4g/L and 6g/L of glucose were added, respectively. Dot Blot and Western Blot were used to identify the expression yields of the different cell pools, and the 5 cell pools with higher expression were selected for the next limiting dilution.
Cells of 5 cell pools selected in the above steps are recovered in a selective culture medium without puromycin and MTX, and limited dilution is carried out when the cell viability reaches more than 90% after 2-5 days. Using a cloning medium (CD FortiCHO medium supplemented with 6mM L-glutamine) at 37 ℃ with CO content of 5% to 8% 2 The static incubator is used for static incubation culture. After three times of screening, 15 clones with higher yield are selected for stability evaluation in the next step.
Resuscitating 15 clones in high yield into 125ml shake flasks containing 8% CO at 37 ℃ 2 Was incubated with a shaker at a rotation speed of 130rpm. Subculturing 1 time every 3 days, and inoculating density of 1.5 × 10 5 Viable cells/ml. Yield of the tracing clone: after subculturing in a new flask, the cells in the old flask were still supplemented with 5g/L glucose, and the flask was incubatedBy day 7, samples were then taken for protein production. And continuously carrying out passage and yield evaluation on the 7 th day, continuously transmitting to 30 generations, and selecting the clone strain with better stability, good cell growth characteristics and higher protein yield for production.
Example 2 preparation of African Swine fever Virus CD2v protein
The CHO cell line selected in example 1 was inoculated into a bioreactor containing Dynamis medium at a density of 3X 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were performed. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. When the cell viability is reduced to about 80%, the cell culture is harvested, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein African swine fever virus CD2v protein is expressed. Protein quantification is carried out according to a BCA protein concentration determination kit method of Biyuntian, and the protein content is 0.5g/L. The CD2v gene without the addition of the signal peptide sequence and Fc sequence was not expressed in CHO cells.
Example 3 preparation of porcine pseudorabies virus gD protein subunit vaccine
With reference to the method of example 1, a CHO cell line that efficiently expresses porcine pseudorabies virus gD protein was constructed, and the gene sequence of porcine pseudorabies virus gD protein is shown in SEQ.ID NO 18. Wherein a signal peptide sequence shown in SEQ ID No.2 is added at the N end of the gD sequence; designing a primer to connect gD added with a signal peptide sequence shown in SEQ ID No.2 and an Fc gene shown in SEQ ID No.15 together through fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Individual viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Sampling every day from day 3, detecting glucose and lactic acid concentration, and performing cell count. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. And culturing until the cell viability is reduced to about 80%, harvesting cell culture, and centrifuging the obtained supernatant to perform Western Blot to confirm that the target protein, namely the porcine pseudorabies virus gD protein is expressed. The content of the target protein is higher, and the content of gD protein is 3g/L.
And (3) uniformly mixing the porcine pseudorabies virus gD protein expressed by the CHO cell strain with an adjuvant 206 according to a proportion, stirring for 15 minutes at the temperature of 30 ℃ at a speed of 120 r/min, and storing at the temperature of 4 ℃ to obtain the porcine pseudorabies virus gD protein subunit vaccine composition. The specific ratio is shown in table 1.
TABLE 1 porcine pseudorabies virus gD protein subunit vaccine ratio
Components Vaccine 1 Vaccine 2
gD protein (μ g/ml) 20 100
206 adjuvant (V/V%) 46 46
Example 4 porcine pseudorabies virus gD protein subunit vaccine immunogenicity assay
12 heads of 21-day-old PRV antigen antibody negative pigletsThe vaccine 1 and the vaccine 2 prepared in example 3 were intramuscularly injected into group 1 and group 2 at an immunization dose of 2 ml/head, and the group 3 control group was injected with 2 ml/head of DMEM medium. 28 days after immunization, porcine Pseudorabies virus HN1201 strain (porcine Pseudorabies virus HN1201 strain, with the preservation number of CCTCC NO. V201311, the preservation unit of China center for type culture preservation, the preservation address of Wuhan university in China, the preservation date of 2013, 5 months and 20 days, and disclosed in China patent application CN 104004774A) is used for virus liquid to attack through intramuscular injection, and the dosage is 2 multiplied by 10 8.0 TCID 50 First, after the challenge, the body temperature of the piglets is measured every day, and clinical symptoms and death conditions are observed. The results are shown in Table 2.
TABLE 2 porcine pseudorabies virus gD protein subunit vaccine immunogenicity test results
Group of Clinical symptoms and death Rate of protection
1 The body temperature is raised for 1-2 days, the appetite is normal, the mental symptoms are basically absent, and the survival is realized 100％(4/4)
2 The body temperature is raised for 1-2 days, the appetite is normal, the mental symptoms are basically absent, and the health food survives 100％(4/4)
3 Has obvious symptoms, 2 deaths after 2 days and all deaths after 3 days 0％(0/4)
The result shows that the porcine pseudorabies virus gD protein subunit vaccine can provide 100 percent (4/4) protection for the piglets after being immunized by the piglets, and the control piglets all die 4 days after being attacked by the virus, thereby showing good immune protection.
EXAMPLE 5 preparation of avian adenovirus Fiber-2 protein subunit vaccine
By referring to the method of example 1, a CHO cell strain capable of efficiently expressing avian adenovirus Fiber-2 protein is constructed, and the gene sequence of avian adenovirus Fiber-2 is shown in SEQ. ID NO 19. Wherein a signal peptide sequence shown in SEQ ID No.3 is added at the N end of the Fiber-2 sequence; primers were designed to join Fiber-2 to which the signal peptide sequence shown in SEQ ID No.3 was added to the Fc gene shown in SEQ ID No.15 by fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were performed. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. When the cell viability is reduced to about 80 percent, the cell culture is harvested, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein, namely the avian adenovirus Fiber-2 protein, is expressed. The content of target protein is high, and the AGP titer of the Fiber-2 protein reaches 1.
Mixing the fowl adenovirus Fiber-2 protein expressed by CHO cell strain and mineral oil adjuvant at a certain proportion, and adding 1% thimerosal solution before stopping stirring to make its final concentration be 0.01%. The specific ratio is shown in Table 3.
TABLE 3 avian adenovirus Fiber-2 protein subunit vaccine ratio
Components Vaccine 3 Vaccine 4
Fiber-2 protein (AGP potency) 1:4 1:32
Mineral oil adjuvant (V/V%) 66％ 66％
EXAMPLE 6 avian adenovirus Fiber-2 protein subunit vaccine immunogenicity assay
30 SPF chickens of 21 days old are divided into 3 groups, each group comprises 10 SPF chickens, the 4 th group to the 5 th group are respectively injected with the vaccine 3 and the vaccine 4 prepared in the immunization example 5 through the neck part subcutaneously, the immunization dose is 0.3ml, and the 6 th group is injected with 0.3ml of physiological saline subcutaneously to be used as a virus attack control. All test chickens were kept separately, and 21 days after immunization, virus liquid was attacked by intramuscular injection and observed for 14 days to record morbidity, mortality and protection number by using FAV-HN strain (fowls adenovirus, FAV-HN strain (bird FAV-HN), with the preservation number of CCTCC NO. V201609, the preservation unit of China center for type culture Collection, the preservation address of university of Wuhan and Wuhan, china, the preservation time of 2016, 2 months and 29 days, disclosed in China patent application CN 107523556A). The results are shown in Table 4.
TABLE 4 avian adenovirus Fiber-2 protein subunit vaccine immunogenicity test results
Figure BDA0003800896740000111
The results show that the 6 th group of the challenge control group is completely killed, and the 4 th to 5 th groups of the immunization groups generate better immune protection to the immunized chickens, so that the immunization effect is good. The poultry adenovirus Fiber-2 protein subunit vaccine prepared by the method can provide effective immune protection for chicken flocks.
Example 7 preparation of avian egg drop syndrome Virus tFiber protein subunit vaccine
By referring to the method of example 1, a CHO cell strain capable of efficiently expressing the tFiber protein of the avian egg-loss syndrome virus is constructed, and the gene sequence of the tFiber protein of the avian egg-loss syndrome virus is shown as SEQ.ID NO 20. Wherein a signal peptide sequence shown in SEQ ID No.4 is added at the N end of the tFiber sequence; primers were designed to link together the tFiber to which the signal peptide sequence shown in SEQ ID No.4 was added and the Fc gene shown in SEQ ID No.15 by fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were made. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. And culturing until the cell viability rate is reduced to about 80%, harvesting the cell culture, and performing Western Blot on the supernatant obtained by centrifugation to confirm that the target protein, namely the poultry egg-loss syndrome virus tFiber protein is expressed. The content of the target protein is high, and the AGP titer of the tFiber protein reaches 1.
Mixing the avian egg drop syndrome virus tFiber protein expressed by the CHO cell strain and the mineral oil adjuvant uniformly according to a proportion, and adding 1% of thimerosal solution before stopping stirring to ensure that the final concentration is 0.01%. The specific mixture ratio is shown in Table 5.
TABLE 5 avian egg drop syndrome Virus Fiber protein subunit vaccine ratios
Components Vaccine 5 Vaccine 6
tFiber protein (AGP potency) 1:8 1:64
Mineral oil adjuvant (V/V%) 66％ 66％
Example 8 avian egg drop syndrome Virus tFiber protein subunit vaccine immunogenicity assay
30 SPF chickens of 21 days old were divided into 3 groups of 10 chickens, and the 7 th to 8 th groups were injected subcutaneously into the neck of each of vaccine 5 and vaccine 6 prepared in immunization example 7, respectively, with an immunization dose of 0.5ml, and the 9 th group was injected subcutaneously with 0.5ml of physiological saline as a blank control. All test chickens were kept separately, and 21 days after immunization, each chicken was bled separately, serum was separated, and serum HI antibody titer of avian egg drop syndrome was determined. The results are shown in Table 6.
TABLE 6 avian egg drop syndrome Virus tFiber protein subunit vaccine immunogenicity test results
Figure BDA0003800896740000121
The results show that the HI antibody titer of the control group 9 at 21 days after immunization is 0, and the HI antibody titers of the immunization groups 7 to 8 are high and the immunization effect is good. The method provided by the invention is used for preparing the vaccine of the protein subunit of the avian egg drop syndrome virus tFiber, and the vaccine can provide effective immune protection for chicken flocks.
EXAMPLE 9 preparation of Chicken infectious bursal disease Virus VP2 protein
Referring to the method of example 1, a CHO cell strain expressing VP2 protein of chicken infectious bursal disease virus was constructed, and the gene sequence of chicken infectious bursal disease virus VP2 was disclosed in Chinese patent application CN103849631A. Wherein a signal peptide sequence shown in SEQ ID No.5 is added at the N end of the VP2 sequence; designing a primer to connect VP2 added with a signal peptide sequence shown in SEQ ID No.5 and an Fc gene shown in SEQ ID No.15 together through fusion.
Inoculating the CHO cell strain to a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were made. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. Culturing until the cell viability rate is reduced to about 80%, harvesting cell culture, centrifuging to obtain supernatant, and performing Western Blot to confirm that the target protein, namely the chicken infectious bursal disease virus VP2 protein, is expressed. The content of the target protein is high, and the AGP titer of the VP2 protein reaches 1.
Example 10 preparation of Leporis Virus VP60 protein
By referring to the method of example 1, a CHO cell strain capable of efficiently expressing the VP60 protein of the Lepidoptera leprosy virus is constructed, and the gene sequence of the VP60 protein of the Lepidoptera leprosy virus is shown in SEQ.ID NO 21. Wherein a signal peptide sequence shown in SEQ ID No.6 is added at the N end of the VP60 sequence; designing a primer to connect VP60 added with a signal peptide sequence shown in SEQ ID No.6 and an Fc gene shown in SEQ ID No.16 together through fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Individual viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were made. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. When the cell viability is reduced to about 80%, cell cultures are harvested, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein rabbit fever virus VP60 protein is expressed. The target protein content is high, and the HA titer of the VP60 protein reaches 16log2.
Example 11 preparation of porcine circovirus type 3 Cap protein subunit vaccine
And (2) constructing a CHO cell strain for efficiently expressing the protein of the type 3 Cap of the porcine circovirus according to the method of the embodiment 1, wherein the gene sequence of the type 3 Cap of the porcine circovirus is shown as SEQ.ID NO 22. Wherein a signal peptide sequence shown in SEQ ID No.7 is added at the N end of the Cap sequence; designing a primer to connect Cap added with a signal peptide sequence shown in SEQ ID No.7 and Fc gene shown in SEQ ID No.15 together through fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were performed. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. And (3) culturing until the cell viability is reduced to about 80%, harvesting cell culture, and carrying out Western Blot on supernatant obtained by centrifugation to confirm that the target protein porcine circovirus type 3 Cap protein is expressed. The content of the target protein is high and is 0.5g/L.
And (3) uniformly mixing the porcine circovirus type 3 Cap protein expressed by the CHO cell strain with a water-soluble adjuvant Gel adjuvant (French Saybord company) in proportion to obtain the porcine circovirus type 3 Cap protein subunit vaccine composition. The specific ratio is shown in Table 7.
TABLE 7 porcine circovirus type 3 Cap protein subunit vaccine ratios
Components Vaccine 7 Vaccine 8
Cap protein (μ g/ml) 25 100
Gel adjuvant (V/V%) 10％ 10％
Example 12 porcine circovirus type 3 Cap protein subunit vaccine immunogenicity assay
15 healthy piglets which are 28-30 days old and have PCV2, PCV3 antigen and antibody negativity detected by ELISA are randomly divided into 3 groups and 5 groups, and the subunit vaccine of the porcine circovirus type 3 Cap protein, which is prepared in the immunization example 11, is immunized. The 10 th to 11 th groups of immune vaccines are 7 to 8 respectively, and the 12 th group of non-immune vaccines are used as a challenge control group. Each immunization group was injected with 2 ml/head of vaccine, and the control group was inoculated with 2 ml/head of physiological saline. The vaccine is attacked 28 days after immunization, the attacking dose is SG strain Porcine Circovirus (Porcine Circovirus type 3 SG), the Porcine Circovirus type 3 SG is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V201712, the preservation date is 3/23 th 2017, the preservation address is university of Wuhan and Wuhan, and is disclosed in China patent application CN 108660115A) 105.0TCID50/head, each piglet is continuously observed after the attacking, and the judgment is carried out according to the clinical symptoms, pathological changes and virus detection results of each piglet, and the specific results are shown in Table 8.
TABLE 8 porcine circovirus type 3 Cap protein subunit vaccine immunogenicity test results
Figure BDA0003800896740000151
The result shows that the porcine circovirus type 3 Cap protein subunit vaccine can provide 100% (5/5) protection for piglets after being immunized by the piglets, and the control piglets are all attacked after being attacked by virus. The porcine circovirus type 3 Cap protein subunit vaccine prepared by the method can provide effective immune protection for swinery.
Example 13 preparation of porcine circovirus type 2 Cap protein
With reference to the method of example 1, a CHO cell line that efficiently expresses the type 2 Cap protein of porcine circovirus was constructed, and the type 2 Cap gene sequence of porcine circovirus was disclosed in Chinese patent application CN101920012A. Wherein a signal peptide sequence shown in SEQ ID No.8 is added at the N end of the Cap sequence; designing a primer, and connecting the Cap added with the signal peptide sequence shown in SEQ ID No.8 and the Fc gene shown in SEQ ID No.15 together through fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Individual viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were performed. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. When the cell viability is reduced to about 80 percent, the cell culture is harvested, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein, namely the porcine circovirus type 2 Cap protein, is expressed. The content of the target protein is high and is 0.6g/L.
EXAMPLE 14 preparation of porcine parvovirus VP2 protein
Referring to the method of example 1, a CHO cell strain capable of efficiently expressing porcine parvovirus VP2 protein is constructed, and the porcine parvovirus VP2 gene sequence is disclosed in Chinese patent application CN103908664A. Wherein a signal peptide sequence shown in SEQ ID No.9 is added at the N end of the VP2 sequence; designing a primer to connect VP2 added with a signal peptide sequence shown in SEQ ID No.9 and an Fc gene shown in SEQ ID No.15 together through fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were made. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. And culturing until the cell viability is reduced to about 80%, harvesting cell culture, and performing Western Blot on supernatant obtained by centrifugation to confirm that the target protein porcine parvovirus VP2 protein is expressed. The content of the target protein is higher and is 2.2g/L.
Example 15 preparation of classical swine fever Virus E2 protein
Referring to the method of example 1, a CHO cell line that can efficiently express classical swine fever virus E2 protein was constructed, and the genetic sequence of classical swine fever virus E2 is disclosed in Chinese patent application CN105527442A. Wherein a signal peptide sequence shown in SEQ ID No.10 is added at the N end of the E2 sequence; designing a primer to connect the E2 added with the signal peptide sequence shown in SEQ ID No.10 and the Fc gene shown in SEQ ID No.15 together through fusion.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were performed. When grapeWhen the sugar level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. When the cell viability is reduced to about 80%, the cell culture is harvested, and Western Blot is carried out on the supernatant obtained by centrifugation to confirm that the target protein classical swine fever virus E2 protein is expressed. The content of the target protein is high and is 2.7g/L.
Example 16 preparation of the schistosoma japonicum GALE protein
Referring to the method of example 1, a CHO cell strain expressing the GALE protein of Schistosoma japonicum with high efficiency was constructed, and the GALE gene sequence of Schistosoma japonicum was disclosed in the Chinese patent application CN102079783A. Wherein a signal peptide sequence shown in SEQ ID No.14 is added at the N end of the GALE sequence; primers were designed to join GALE to which a signal peptide sequence shown in SEQ ID No.14 was added and the Fc gene shown in SEQ ID No.16 by fusion. In this example, the signal peptide sequence shown in SEQ ID No.14 was used, but any of the other signal peptide sequences shown in SEQ ID No.1-13 may be used. This also applies to the other embodiments. The Fc gene sequences shown in SEQ ID No.15 and SEQ ID No.16 can also optionally be used in the various embodiments.
Inoculating the selected CHO cell strain into a bioreactor containing Dynamis culture medium at a density of 3 × 10 5 Viable cells/ml. The parameters are set to be pH7.1-7.2, dissolved oxygen is 40%, the temperature is 37 ℃, and the stirring speed is 130rpm. Samples were taken daily from day 3, glucose and lactate concentrations were measured, and cell counts were performed. When the glucose level was below 2g/L, glucose was fed to 6g/L.
Meanwhile, 1 XCD efficiency C + AGT additive is added on the 3 rd day, the 5 th day, the 7 th day and the 10 th day after inoculation, and the adding amount is 10 percent of the volume of the culture solution each time. Culturing until the cell viability is reduced to about 80%, harvesting cell culture, centrifuging to obtain supernatant, and performing Western Blot to confirm that the target protein schistosoma japonicum GALE protein is expressed. The content of the target protein is higher and is 1.2g/L.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A method for constructing a CHO cell expressing a foreign protein, wherein the method comprises: adding a signal peptide sequence at the N end of an exogenous protein sequence; step (2) the foreign protein sequence added with the signal peptide sequence is fused with an Fc sequence to construct an expression plasmid; step (3) transfecting CHO cells with the expression plasmid constructed in the step (2); and (4) harvesting the exogenous protein.
2. The method of claim 1, wherein the signal peptide sequence in step (1) is as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No. 14.
3. The method according to claim 1 or 2, wherein the Fc sequence in step (2) is shown as SEQ ID No.15 or SEQ ID No. 16.
4. The method according to claim 1, wherein the foreign protein gene in the step (1) comprises African swine fever virus CD2v protein, avian adenovirus Penton protein, avian adenovirus Fiber-2 protein, avian egg-loss syndrome virus Penton protein, avian egg-loss syndrome virus tFiber protein, chicken infectious bursal disease virus VP2 protein, porcine circovirus type 3 Cap protein, porcine circovirus type 2 Cap protein, porcine pseudorabies virus gB protein, porcine pseudorabies virus gD protein, porcine parvovirus VP2 protein, porcine pestivirus E2 protein, bovine infectious rhinotracheitis virus gB protein, bovine infectious rhinotracheitis virus gD protein, foot and mouth disease virus VP0 protein, foot and mouth disease virus VP3 protein, foot and mouth disease virus VP1 protein, rabbit plague virus VP60 protein, japanese schistosome GALE protein, japanese schistosome Wnt5 protein.
5. A CHO cell expressing a foreign protein constructed by the method of any one of claims 1 to 4.
6. A method of expressing a foreign protein, wherein the method uses the CHO cell of claim 5 to express the foreign protein.
7. Use of a CHO cell according to claim 5 or the method according to claim 6 for preparing a foreign protein.
8. A vaccine composition, wherein the vaccine composition comprises a foreign protein prepared using the method of claim 6, and a pharmaceutically acceptable carrier; in particular, the vaccine composition is a subunit vaccine composition.
9. The vaccine composition of claim 8, wherein the pharmaceutically acceptable carrier comprises an adjuvant comprising: (1) Mineral oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block copolymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, montanide ISA 206, and Gel adjuvant; preferably, the saponin is QuilA, QS-21, GPI-0100;
preferably, the content of the adjuvant is 5% -70% > -V/V;
preferably, the content of the adjuvant is selected from 5%/V, 6%/V, 7%/V, 8%/V, 9%/V, 10%/V, 15%/V, 20%/V, 25%/V, 30%/V, 35%/V, 40%/V, 45%/V, 50%/V, 55%/V, 60%/V, 65%/V, 66%/V, 67% V/V, 70% V/V;
optionally, the vaccine composition further comprises one or more of the group consisting of: drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives; the immunostimulant is, for example, interferon-alpha, interferon-beta, interferon-gamma, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor and interleukin 2.
10. The vaccine composition of claim 8, wherein the vaccine composition is an oral dosage form or a non-oral dosage form;
preferably, the vaccine composition is a non-oral dosage form administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.
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