CN115948469B - A method for increasing the secretion rate of foreign protein expressed in silkworm cells - Google Patents

Abstract

A method for improving the secretion rate of foreign proteins expressed in silkworm cells, extracting cDNA obtained by reverse transcription of all RNA of silkworm BmN cells, synthesizing spz gene by using the cDNA obtained by reverse transcription as a template, and then transferring the spz gene shown in SEQ ID NO.1 into silkworm BmN cells, which can improve the secretion rate of foreign proteins expressed in silkworm cells. The present invention can effectively improve the secretion efficiency of foreign proteins expressed in silkworm cells, provide a new research direction and idea for improving the secretion rate of foreign proteins expressed in silkworm cells, and provide a reference for the study of the mechanism of signal peptides involved in protein secretion.

Description

Method for improving secretion rate of foreign protein expressed by silkworm cells

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving secretion rate of foreign proteins expressed by silkworm cells.

Background

Along with the improvement of human living standard and the enhancement of health consciousness, the demands of various recombinant protein medicines for treatment or health care are rising year by year, and meanwhile, the production value of biotechnology industry for producing vaccine, immunoglobulin, antibody, protein factor and other recombinant proteins for treatment is increased in speed, so that the status in the biomedical industry is also more and more prominent. Therefore, in recent years, studies on increasing the expression level of recombinant proteins, particularly secreted proteins and membrane proteins whose expression level is low, have been attracting attention.

Many proteins need to be secreted extracellularly or localized on membranes to exert their biological functions, and many studies have shown that membrane proteins and secreted proteins have low expression efficiency (S Chakrabarti,M Robert-Guroff,F Wong-Staal,et al.Expression of the HTLV-III envelope gene by a recombinant vaccinia virus.1986 Nature.320(6062):535-7.doi:10.1038/320535a0.).. In recent years, there are many studies and patents on improving the expression of secreted proteins in yeast cells, insect cells and mammalian cells. Zhang Xinran et al disclose a method for improving the expression level of secretory foreign proteins of Pichia pastoris by coexpression of double genes of HAC1 and ERO1 or three genes of HAC1, ERO1 and BIP in Pichia pastoris cells, so as to effectively improve the expression level of secretory foreign proteins of Pichia pastoris [ ZL2014103955743]. There are also studies and reports that the expression and secretion rate [De Pinheiro C G M,Pedrosa M d O,Teixeira N C,et al.,2016.Optimization of canine interleukin-12 production using a bacμLovirus insect cell expression system.BMC Res.Wang B-Z,Liu W,Kang S-M,et al.,2007.Incorporation of High Levels of Chimeric Human Immunodeficiency Virus Envelope Glycoproteins into Virus-Like Particles.J.Virol.81(20)]. of membrane proteins can be greatly improved by utilizing signal peptide sequences of proteins which effectively target the endoplasmic reticulum of insect cells, such as the alfalfa silver vein moth nuclear polyhedrosis virus (AcMNPV) GP64 protein or bee toxin signal peptide, and the like, the silkworm has a long feeding history in China, and the silkworm larvae are large and have no flying ability and can be fed aseptically, so that the recombinant protein is suitable for being used as a biological factory to produce recombinant proteins. Since Smith et al expressed interferon in silkworm cells in 1983, thousands of proteins have been expressed in silkworm cells, larvae and pupae. Japanese scholars R Fujita and the like successfully express S protein [Ryosuke Fujita,Masato Hino,Takeru Ebihara,et al.Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.2020.Biochem Biophys Res Commun,529(2):257-262.doi:10.1016.], of secretory SARS-CoV-2 in silkworm blood stranguria in 2020, and provide theoretical information for further application to preparation of detection kits, vaccines and the like of new coronaviruses. If the secretion amount of the secreted protein can be increased, the effect of reducing the protein expression cost is achieved, and the cost of the protein applied to various aspects such as medicines, antigen preparation, detection kits and the like is further reduced.

Disclosure of Invention

The invention discloses a method for improving secretion rate of foreign proteins expressed by silkworm cells, which utilizes cDNA synthesized by reverse transcription of total RNA of silkworm BmN cells as a template to synthesize spz genes, constructs pIZ/V5-spz recombinant plasmid, and can obviously improve secretion rate of foreign proteins in the cells after transfection of BmN cells for 72 hours.

The technical scheme is that spz gene shown in SEQ ID NO.1 is applied to improving secretion rate of foreign protein expressed by silkworm cells.

The cell line for stably expressing spz gene shown in SEQ ID NO.1 is applied to improving secretion rate of exogenous protein expressed by silkworm cells.

The application of the baculovirus vector for stably expressing spz gene shown in SEQ ID NO.1 in improving the secretion rate of exogenous protein expressed by silkworm cells.

A method for improving secretion rate of foreign protein expressed by silkworm cells comprises extracting cDNA obtained by reverse transcription of all RNA of silkworm BmN cells, synthesizing spz genes by taking the cDNA obtained by reverse transcription as a template, and transferring spz genes shown in SEQ ID NO.1 into the silkworm BmN cells.

The invention has the beneficial effects that the invention discovers that the total RNA of BmN cells is extracted and reversely transcribed to obtain cDNA, the spz gene synthesized by taking the cDNA as a template and the pIZ/V5 vector construct the instantaneous expression recombinant plasmid and the BmN cells are transfected, and the secretion rate of the exogenous protein can be obviously improved after 72 hours. The method is simple to operate, low in cost and easy to popularize, and can remarkably improve the secretion efficiency of the exogenous protein expressed by the silkworm cells. The invention can effectively improve the secretion efficiency of the expressed exogenous protein in the silkworm cells, provides a new research direction and thought for improving the secretion rate of the expressed exogenous protein in the silkworm cells, and provides a reference for the research of the mechanism of the signal peptide participating in protein secretion. The method provided by the invention is simple and clear, easy to understand, simple and feasible to operate and high in efficiency.

Drawings

FIG. 1 shows the total luciferase expression level after a stable cell line was selected by transfecting BmN cells with pIZ/V5-SP-Luc and pIZ/V5-Luc, transferring the recombinant plasmid pIZ/V5-spz into BmN cells, and comparing with pIZ/V5 empty cells for 72 hours.

FIG. 2 shows the secretion of the supernatant after a further 72h after the selection of stable cell lines by transfecting BmN cells with pIZ/V5-SP-Luc and pIZ/V5-Luc and transferring the recombinant plasmid pIZ/V5-spz into BmN cells, with no load of pIZ/V5 as a control.

FIG. 3 shows the secretion rate after 72 hours calculated using the supernatant secretion amount and the total expression amount.

Detailed Description

Liu Na et al show that (18Additional Amino Acids of the Signal Peptide of the Bombyx mori Nucleopolyhedrovirus GP64 Activates Immunoglobulin Binding Protein(BiP)Expression by RNA seq Analysis[J].Current Microbiology,2020.doi:10.1007/s00284-020-02309-4.), transfects siRNA targeting BiP in BmN cells, and by taking a randomly disturbed siRNA sequence (NC) as a control, extracting RNA after 24 hours, and detecting the interference efficiency of BiP by using qPCR technology after reverse transcription, the result shows that the siRNA remarkably reduces the expression level of BiP in host cells. And (3) continuing to transfect pIZ/V5-SP-Luc after determining that BiP in a host cell is effectively interfered, taking an empty vector pIZ/V5-Luc as a control group, respectively collecting supernatant and cell samples after 72 hours, and fully lysing the samples by using cell lysate to detect the luciferase activity. The results show that the protein secretion rate is significantly increased compared with the control group after the BiP interference.

Example 1

1. Cell lines and plasmids

BmN cells were maintained by a laboratory of major genetic improvement of silkworms in agricultural rural areas and cultured at 27℃on the basis of TC-100 culture with 10% fetal bovine serum (Thermo FISHER SCIENTIFIC).

In order to facilitate statistics of the secretion rate, a luciferase reporter gene is fused with a signal peptide (SIGNAL PEPTIDE, SP) of the silkworm nuclear polyhedrosis virus GP64 to construct a recombinant plasmid pIZ/V5-SP-Luc, and pIZ/V5-Luc is used as a control (after BmN cells are transfected by reference ：Na Liu,Jinshan Huang,Lin Liu,Frank Boadi,et al.,2021,18Additional Amino Acids of the Signal Peptide of the Bombyx mori Nucleopolyhedrovirus GP64 Activates ImmunoglobμLin Binding Protein(BiP)Expression by RNA-seq Analysis.Current Microbiology.78:490–501.), in the construction process, clone cell lines for stably expressing the SP-Luc and the Luc are screened).

2. Construction of transient expression vector carrying Spz Gene

1) Collecting BmN cells in logarithmic growth phase, and extracting total RNA of the cells, wherein the specific operation is as follows:

① Removing the culture medium, adding 2mL of PBS for suction washing, centrifuging at 4000rpm for 10min, discarding the supernatant, adding 1mL of TRIZOL, and suspending;

② Adding 200 mu L of chloroform, manually shaking vigorously, shaking evenly until the color of the milk becomes pink, standing on ice for 2-3 min, centrifuging at 12000rpm and 4 ℃ for 15min;

③ Sucking the supernatant, adding equal volume of isopropanol, mixing (mixing upside down, incubating for 10min on ice, centrifuging at 12000rpm and 4 ℃ for 15min, and discarding the supernatant;

④ Adding 75% ethanol-DEPC with volume of 2 times, mixing, centrifuging at 7500rpm for 5min, and discarding supernatant;

⑤ Repeating the step (4) once;

⑥ Drying the super clean bench at room temperature for 5min

⑦ And adding 20-50 mu LRNaes-free ddH ₂ O, measuring concentration, and detecting whether the extraction is correct or not by running gel (taking 5 mu L,180V,7 min).

2) The cDNA is synthesized by reverse transcription, and the specific procedures are as follows:

① Removal of genomic DNA

The following systems were placed in RNase-free centrifuge tubes

Gently sucking and beating the mixture by a pipette, mixing the mixture evenly, standing the mixture for 2 minutes at the temperature of 42 ℃.

② Preparation of reverse transcription reaction System

16. Mu.L of the reaction solution was left in the ① reaction tube, 4. Mu.L of 5X HISRIPTIIIQRT SUPER MIX was added thereto, and the mixture was pipetted and homogenized.

③ Reverse transcription reaction

The reaction was carried out at 37℃for 15min, after which time the reaction was carried out at 85℃for 5s. -20 ℃ for subsequent PCR reactions.

3) PCR amplification spz gene and cloning the amplified gene to pIZ/V5-His vector and enzyme digestion identification

A pair of primers was designed, the sequence was as follows:

F:CGCGAATTC ATGTCACTTATACTAAGAGCTTT

protective base EcoRI Tm at 60℃and GC content 30%

R:GCGTCTAGA CTAAGTATGGTTTGATTCGATG

The protected base XbaI Tm is 60 ℃ and the GC content is 36%

And (3) using the reverse transcription synthesized cDNA as a template, amplifying Spz genes by PCR, cloning the amplified genes to a pIZ/V5-His vector, and performing enzyme digestion and identification to be correct for later use.

The PCR amplification of spz gene includes setting reaction conditions of 98-5 min, 98-10 s, 55-15 s, 72-10 s and 72-7 min for 35 cycles. The system is as follows:

And (3) identifying PCR products, namely preparing glue, adding 5 mu L of 10 multiplied by loding buffer into 50 mu L of the product, uniformly mixing, fully spotting, adding 5 mu LDL2000 maker and 120V, running the glue for 20min, and identifying that the glue is recovered correctly.

And (3) recycling PCR product glue:

① Placing the gel block containing the target fragment cut from the agarose gel into an EP tube;

② Adding 500 mu L of Buffer B2, and carrying out sol in a water bath at 50 ℃ for 5-10 min;

③ Transferring the sol into an adsorption column, centrifuging at 8000xg for 1min, and pouring out the liquid in a collecting pipe;

④ Adding 500 μL of Wash Solution, centrifuging at 9000xg for 1min, pouring out the liquid in the collection tube, repeating for one time

⑤ The empty adsorption column 9000xg is centrifuged for 1min;

⑥ Placing the adsorption column into a new 1.5mL centrifuge tube, standing for 5min, adding 30 μL of absorption Buffer in the center of the adsorption film, standing for 1min at room temperature, centrifuging for 1min, and preserving the DNA solution in the tube. The concentration was found to be 98.7 ng/. Mu.L (OD 260/280=1.86).

Spz and pIZ/V5:

spz enzyme cutting system

PIZ/V5 cleavage system

SPZ digestion conditions are 37 ℃ water bath for 15min, digestion products are directly recovered (see the specification of a PCR product recovery kit for details), and the concentration is measured to be 20.6 ng/. Mu.L after recovery.

The pIZ/V5 plasmid was digested in a 37℃water bath for 15min, run-out identification and recovery (130V,15min,DL15000 maker) was performed, and the concentration was found to be 7.3 ng/. Mu.L after recovery. After recovery, the connection was performed for 10min at 25 ℃, the connection system was as follows:

(note: spz to carrier material amount (n) should satisfy a ratio of 4:1)

Conversion of ligation products:

① Adding 50-100 mu L of competent cells into the connection product (precooling), and standing on ice for 30min;

② Heat shock is carried out for 90s in a 42 ℃ water bath, and the mixture is immediately placed on ice for standing for 2min;

③ Adding 800 mu L of LB culture medium, and resuscitating for 1h by a shaking table;

④ Centrifuging the resuscitated bacteria at 7000rpm for 2min;

⑤ Removing the supernatant to 100 mu L, sucking and suspending, and coating a plate containing solid LB culture medium (containing zeocin antibiotics);

⑥ Culturing in a 37 ℃ incubator.

Plasmid extraction Single colonies were picked in 4mL LB liquid medium, added with 0.8. Mu.L zeocin, and shaken overnight.

① Taking 1.5mL of fungus liquid cultured overnight, centrifuging 8000 Xg for 2min, collecting fungus body, and discarding the culture medium;

② 250 mu L Buffer P1 is added into the sediment to thoroughly suspend the thalli;

③ Adding 250 mu L Buffer P2, immediately and gently reversing the centrifuge tube for 5-10 times, uniformly mixing, and standing at room temperature for 2-4 min;

④ Adding 350 mu L Buffer P3, immediately and gently reversing the centrifuge tube for 5-10 times and uniformly mixing;

⑤ Centrifuging at 12000 Xg for 10min, transferring the supernatant to adsorption column, centrifuging at 8000 Xg for 1min, and pouring out the liquid in the collecting tube;

⑥ Adding 500 μl of Wash Solution, centrifuging 9000×g for 1min, and pouring out the liquid in the collection tube;

⑦ Repeating step ⑥ once;

⑧ Centrifuging the empty adsorption column in 9000 Xg for 1min;

⑨ The column was placed in a clean 1.5mL centrifuge tube, allowed to stand for 5min, 50. Mu.L of the absorption Buffer was added to the center of the absorption membrane, and the DNA solution was allowed to stand at room temperature.

Enzyme digestion identification, wherein the enzyme digestion is carried out for 15min at 37 ℃, and the enzyme digestion system is as follows:

after completion of the cleavage, the gel was run for identification, 5. Mu.L was sampled, DL5000 maker was used as a reference (5. Mu.L), 130V,15min.

3. Screening BmN clone cell line for stably expressing SP-Luc and Luc

See the description for details with reference to Invitrogen ^TM pIZ/V5-His vector kit (Thermofiser. Cn), the steps are as follows:

① BmN cells were maintained in a laboratory with improved emphasis on silkworm genetics in rural agricultural sectors, and cultured with TC-100 containing 10% fetal bovine serum (Thermo FISHER SCIENTIFIC) based on 27℃culture

② Fusing a luciferase reporter gene and a Signal Peptide (SP) to construct a recombinant plasmid pIZ/V5-SP-Luc, and taking pIZ/V5-Luc as a control;

③ Respectively transfecting the recombinant plasmids into BmN cells;

④ 48h after transfection, the transfection solution was removed and fresh medium was replaced (no Zeocin ^TM);

⑤ Dividing the cells 1:5 and allowing the cells to attach for 15min, then replacing the cells with Zeocin ^TM medium with proper concentration, culturing the cells at 27 ℃, replacing the selective medium every 3-4 days, and screening for 60 days;

⑥ The stably expressed clonal cell lines were isolated and assayed for luciferase expression.

Transfection of two BmN cell lines with pIZ/V5-spz, respectively

Two BmN clone cell lines were respectively transfected with 2. Mu.g of recombinant plasmid pIZ/V5-spz, and 2. Mu.g of pIZ/V5 empty cells were used as a control, and after 72 hours, the transfected cells and cell culture medium were collected, respectively, and luciferase activity was measured.

The transfection procedure was as follows:

① Cells were seeded one day in advance in 24-well plates, preferably at a cell density of about 60% at the time of transfection;

② Diluting pIZ/V5-spz of 2 μg with 25 μl serum-free solution, and mixing completely to obtain DNA diluent;

③ mu.L of ENTRANSTER ^TM -H4000 was diluted with 25. Mu.L of serum-free diluent and thoroughly mixed to prepare ENTRANSTER ^TM -H4000 diluent which was allowed to stand at room temperature for 5min.

④ And respectively adding ENTRANSTER ^TM -H4000 diluent into pIZ/V5-spz diluent, fully and uniformly mixing (sucking and blowing by a sample feeder for 20 times), and standing for 15min at room temperature. The preparation of the transfection complex is completed.

⑤ Mu.L of the transfection complex was added to a culture vessel containing cells and complete medium, and gently mixed.

⑥ After 4 hours of culture, the medium was changed, and the culture was routinely performed for 72 hours to determine the relative activity of luciferase.

Influence of pIZ/V5-spz on the expression level and secretion rate of foreign proteins after transfection of BmN cells

The collected cells and medium samples were assayed for luciferase activity as follows:

(1) Diluting the 5 x cell lysate with PBS to a1 x cell lysate;

(2) The medium in the 24-well plate of the above experiment was aspirated into a new EP tube of the corresponding number;

(3) 120 μl of 1×cell lysate was added to each well of the 24-well plate, and the incubator was incubated with shaking for 15min;

(4) Sucking the cell lysate in the well into another new EP tube of the corresponding number with a pipette;

(5) Centrifuging 12000 Xg of all the sample EP tubes filled with the culture medium and the cell lysate for 2min, taking 4 mu L of each EP tube in a new corresponding EP tube, and continuing 12000 centrifugation for 2min;

(6) The instrument detects and records receipts, counts the total expression amount of luciferase and the amount secreted into the culture medium, and calculates the secretion rate.

The statistical analysis results were:

the statistics of the total expression amount, the amount secreted into the culture medium and the secretion rate are shown in the following figures 1-3 respectively;

Screening stable cell lines for stably expressing SP-Luc and Luc, transferring 2 mug of recombinant plasmid pIZ/V5-spz into BmN cells, taking pIZ/V5 empty load as a control, collecting cells and supernatant after 72 hours, measuring luciferase activity, and calculating secretion rate. The results showed that in BmN expressing secreted protein (SP-Luc), the secretion amount of SP-Luc supernatant was significantly increased after transfection pIZ/V5-spz, after 72h of culture, relative to pIZ/V5 no-load control group (FIG. 2). The experimental results showed an increase in secretion rate of 12.7% (fig. 3).

Finally, it should be understood that the spz amounts, modes of use and times of treatment described in the examples and embodiments above are for illustrative purposes only and are not intended to be limiting, and those of ordinary skill in the art will appreciate that various changes in form and detail may be made thereto, or that the effects described in this patent may be similarly achieved, for example, by constructing a stable cell line that overexpresses the spz gene, or by carrying the spz gene using a vector such as a baculovirus, without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (2)

1. Application of the spz gene shown in SEQ ID NO.1 in increasing the secretion rate of foreign proteins expressed with the participation of the signal peptide of Bombyx mori nuclear polyhedrosis virus GP64 in Bombyx mori BmN cells. 2. Application of a baculovirus vector stably expressing the spz gene shown in SEQ ID NO.1 in increasing the secretion rate of foreign proteins expressed with the participation of the signal peptide of Bombyx mori nuclear polyhedrosis virus GP64 in Bombyx mori BmN cells.