CN114958757B - Recombinant ST cell capable of stably expressing TreT, construction method and application thereof - Google Patents
Recombinant ST cell capable of stably expressing TreT, construction method and application thereof Download PDFInfo
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 35
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a recombinant ST cell for stably expressing trehalose transport protein, and a construction method and application thereof. Recombinant ST cells capable of stably expressing trehalose transport proteins are obtained through a bioengineering means, and then ST cells with trehalose accumulation capacity are obtained through a way of adding trehalose into a culture environment. The cell can protect the virus in the process of propagating the progeny virus, and experiments prove that the recombinant ST cell propagates the virus, so that the survival rate of the virus is obviously improved at 37 ℃, and the obvious maintenance effect on the survival rate of the virus is shown for different freezing conditions.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant ST cell for stably expressing trehalose transport protein, and a construction method and application thereof.
Background
Trehalose is a non-reducing disaccharide, and is formed by connecting two molecules of glucopyranose through alpha, alpha-1, 1 glycosidic bonds. Has stable chemical property. Trehalose can provide effective protection for biological macromolecules in extreme environments such as high temperature, low temperature, high osmotic pressure, drying and the like.
The midge (Polypedilum vanderplanki) larvae can accumulate a large amount of trehalose under extreme environments such as desiccation or salt stress, so that the activity is recovered after the larvae are separated from the extreme environments. Thus, the aquatic midge is also known as "dehydrated organism". The reason for this is that the chironomus aquatic larvae can express trehalose transport proteins (Trehalose transporter, treT) in extreme environments, thereby transporting trehalose into the body and achieving accumulation of trehalose.
ST cells, porcine testicular cells (swovestis), are widely used in the proliferation of enveloped viruses.
The ST cells with the capacity of synthesizing or transporting trehalose are obtained through biotechnology means, so that the survival rate of progeny viruses in a low-temperature freezing environment is improved, and the ST cells have positive effects and important significance for products based on viruses.
Thus, finding an ST cell capable of synthesizing or transporting trehalose is a research hotspot for those skilled in the art. However, there is no report on the synthesis and transport of trehalose by mammals, and this is a difficulty for those skilled in the art.
Disclosure of Invention
Aiming at the report about the synthesis and transport of trehalose in mammals at present, the invention discloses a recombinant ST cell capable of stably expressing trehalose transport protein, a construction method of the cell and application of the cell in virus proliferation, thereby being expected to realize the transport and accumulation of trehalose in a culture environment into cells by animal cells.
Specifically, the invention discloses a recombinant ST cell (ST-Sus-Tret-1) capable of stably expressing trehalose transport protein, which is preserved in China general microbiological culture Collection center (CGMCC) at 11/6/2020, with the preservation number of CGMCC No.21010 and the preservation address of China academy of microorganisms at 3 of North Chenxi Lu No.1 in the Korean region of Beijing.
The recombinant ST cells contain a trehalose transport protein coding sequence Tret-1 shown in SEQ ID 1, wherein the trehalose transport protein coding sequence Tret-1 is a recombinant nucleotide sequence optimized according to pig codon preference.
Meanwhile, the invention also discloses a construction method of the recombinant ST cells, which comprises the following steps:
(1) Constructing a vector pCMV-Tret-1 containing a trehalose transporter coding sequence;
(2) The transfection complexes were added drop wise to ST cell culture broth, transfected, and G418 was used to screen for stable and continuously growing recombinant ST-Sus-Tret-1 cells in resistance.
Wherein, the ST cells are ST-Sus cells grown in single cell suspension.
Preferably, in the step (2), two rounds of screening are performed using G418, wherein the screening concentration in the first round is 500ug/ml, and the screening concentration in the second round is 1000ug/ml. Preferably, 20 successive rounds of screening are used.
Furthermore, the invention also discloses the application of the recombinant ST cells (ST-Sus-Tret-1) in proliferating viruses.
Specifically, in the invention, the target virus to be proliferated is further subjected to single cell clone screening on recombinant ST cells (ST-Sus-Tret-1), so that single cell clones aiming at the target virus are obtained, and the target virus is used for proliferation of the virus.
In a preferred embodiment, the recombinant ST cells ST-Sus-Tret-1 are used for the proliferation of Porcine Epidemic Diarrhea Virus (PEDV).
Furthermore, the invention also discloses a monoclonal ST-Sus-Tret-1-9E2 for proliferation of Porcine Epidemic Diarrhea Virus (PEDV).
In a preferred embodiment, the recombinant ST cells (ST-Sus-Tret-1) are used for the proliferation of Classical Swine Fever Virus (CSFV).
Furthermore, the invention also discloses a single cell clone ST-Sus-Tret-1-4C7 for proliferating Classical Swine Fever Virus (CSFV).
In a preferred embodiment, the recombinant ST cells (ST-Sus-Tret-1) are used for the proliferation of pseudorabies virus (PRV).
Furthermore, the invention also discloses a single cell clone ST-Sus-Tret-1-3D3 for propagating pseudorabies virus (PRV).
The invention obtains the recombinant ST cells capable of stably expressing the trehalose transport protein through a bioengineering means, and further obtains ST cells with trehalose accumulation capacity through adding trehalose into a culture environment. The cell can protect the virus in the process of propagating the progeny virus, and experiments prove that the recombinant ST cell propagates the virus, not only can the survival rate of the virus be obviously improved under the condition of 37 ℃, but also the survival rate of the virus can be obviously maintained under different freezing conditions.
Drawings
FIG. 1 is an electrophoresis chart of the enzyme digestion of a Tret-1 recombinant expression vector;
wherein M: DNA marker,1:pCMV-Tret-1 was digested with NheI and NotI.
FIG. 2 shows the detection of recombinant expression of Tret-1 by a cell clone recovering adherent growth after FACS sorting of ST-Sus-Tret-1 cells.
Detailed Description
For a better understanding of the present invention, we will further describe the present invention with reference to specific examples.
EXAMPLE 1 construction of ST-Sus-Tret-1 cells
1.1 vector construction
The nucleotide sequence codon of the trehalose transport protein (Tret) is optimized according to the pig codon preference, and the optimized recombinant nucleotide sequence Tret-1 is shown as SEQ ID NO. 1. The sequence was cloned into pUC57 vector to give pUC-Tret-1 vector.
The fragment is amplified from pUC-Tret-1 plasmid containing Tret-1 coding sequence by using upstream and downstream primers with NheI and NotI cleavage sites, and cloned into eukaryotic expression vector of same double cleavage after double cleavage, so as to construct eukaryotic expression vector pCMV-Tret-1 of Tret-1 for transfection operation.
Specifically, the primers p1, p2 used in this example are as follows:
p1：GCCGGTACCATGGAGCTGAACAACAAGGAGGAC (NheI cleavage site at the streak)
p2：GCCGCGGCCGCTCACTTGTCATCGTCGTCCTTG (NotI cleavage site at the streaked position)
The pUC-Tret-1 is used as a template, the coding sequence of the Tret-1 is amplified by PCR reaction, and a flag label is added at the C end. The target fragment amplified by PCR and the pCMV plasmid are treated by NheI and NotI double enzyme digestion, and a recombinant eukaryotic expression vector pCMV-Tret-1 is constructed, and the enzyme digestion identification is shown in figure 1. The plasmid for transfection with higher quality is obtained by extraction with Qiagen company plasmid big extraction kit.
1.2 production of ST-Sus-Tret-1 cells
The pCMV-Tret-1 is transfected into ST-Sus cells grown in single cell suspension, and the recombinant ST cells with resistance to G418 drugs and capable of stably expressing the Tret-1 protein are obtained by two rounds of screening with the G418 drugs.
Specifically, ST-Sus cells were first grown at an initial cell density of 5X 10 5 cells/ml were inoculated into 50ml Falcon tubes at 15ml,180rpm,37℃and 5% CO 2 After overnight incubation, used for transfection.
The medium was replaced with Opti-MEM medium prior to transfection and incubated in the incubator for 10 minutes.
The transfection complex was then formulated according to the instructions of the 25kDa branched PEI transfection reagent and incubated for 5 minutes at room temperature. After incubation is completed, the transfection complex is added drop-wise to ST-Sus cells, placed in an incubator for 6-8 hours, during which the cells are intermittently agitated.
After the transfection was completed, the culture supernatant containing the remaining transfection complex was removed and replaced with a normal medium for suspension culture.
ST-Sus cells 24-72 hours after transfection were re-inoculated in a 1:3-1:5 ratio into 50ml Falcon tubes at 180rpm,37℃at 5% CO 2 Recombinant cell selection was performed in G418 medium containing 500 ug/ml. G418 was performed at a frequency of 3 days for the primary fluidThe screening was continued for a first round of screening time of approximately 2 to 3 weeks (20 consecutive generations). Recombinant ST-Sus-Tret-1 cells capable of sustained proliferation under G418 selection pressure were selected for continued culture. In the second round of screening, the working concentration of G418 screening is increased to 1000ug/ml, and the pressure screening is continued for 20 generations according to the screening method of the first round, so that the recombinant ST-Sus-Tret-1 cells which can stably and continuously grow in the G418 screening resistance are finally obtained.
EXAMPLE 2 preparation of monoclonal ST-Sus-Tret-1-9E2, monoclonal ST-Sus-Tret-1-4C7 and monoclonal ST-Sus-Tret-1-3D3
Screening recombinant ST-Sus-Tret-1 cells which stably and continuously grow in the resistance obtained in the example 1, sorting the cells into a 96-well plate by a flow cytometer to form single cell clones in each well, and preparing cell clone double plates after the cells recover to adherent growth to form 173 cell clones which effectively adherent grow, wherein the detection of the recombinant expression of the Tret-1 of the cell clones is shown in figure 2.
Aiming at PEDV, CSFV, PRV proliferation, screening single cell clones by adopting PEDV, CSFV and PRV, screening cell clones which are optimal for proliferation of the three viruses from 173 cell clones, and naming the cell clones respectively to obtain single cell clone ST-Sus-Tret-1-9E2 which is high-efficiency proliferation of the PEDV, single cell clone ST-Sus-Tret-1-4C7 which is high-efficiency proliferation of the CSFV and single cell clone ST-Sus-Tret-1-3D3 which is high-efficiency proliferation of the PRV.
EXAMPLE 3 use of single cell suspension grown ST-Sus-Tret-1-9E2 cells in proliferation of PEDV
ST-Sus-Tret-1-9E2 cells were plated at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells.
To the viable cell density of 4.5X10 6 The cells/ml and PEDV receives virus according to MOI=0.1, the virus proliferation period is 24-36 hours, and the virus titer reaches 8.75-9.0 lgTCID 50 /ml。
Example 4 comparison of the effects of ST-Sus-Tret-1-9E2 cells with Primary ST-Sus cells in PEDV proliferation applications
4.1 ST-Sus-Tret-1-9E2 cells were expressed as 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 10×10 6 PEDV was inoculated at a rate of moi=0.3, and the virus growth was carried out by increasing the volume of the virus growth maintenance solution (containing trehalose 0.1 to 80 um) by 1 time, with the growth cycle being 24 to 36 hours.
The living cells of ST-Sus-Tret-1-9E2 grown by single cell suspension are reduced to 0.1-1.2X10 6 Virus harvest can be performed at cells/ml.
The harvested virus can be preserved at 4-70 ℃ for 1 year. The proliferation potency of PEDV can reach 8.75-9.0 lgTCID 50 Per ml, the loss of viral potency after one year of storage at-20 ℃ is less than 0.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is less than 0.2lgTCID 50 /ml。
4.2 the original ST-Sus cells were grown at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 10×10 6 PEDV was inoculated at a rate of moi=0.3, and the virus growth was carried out by increasing the volume of the virus growth maintenance solution (containing trehalose 0.1 to 80 um) by 1 time, with the growth cycle being 24 to 36 hours.
The living cells of ST-Sus grown in single cell suspension are reduced to 0.1-1.2X10 6 Virus harvest can be performed at cells/ml.
The proliferation potency of the original ST-Sus cells is 7.33-7.77 lgTCID 50 Per ml, the loss of viral potency after one year of storage at-20℃is greater than 2.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is greater than 1.5lgTCID 50 /ml。
EXAMPLE 5 use of single cell suspension grown ST-Sus-Tret-1-4C7 cells in the proliferation of CSFV
ST-Sus-Tret-1-4C7 cells were plated at 1X 10 6 Single cell suspension culture is carried out by taking cells/ml as initial culture density, and the density of living cells reaches 2.5X10 6 The cells/ml, CSFV receives virus according to 2% (volume ratio), the virus proliferation period is 13-15 days, the virus liquid is harvested in 3-5 batches, and the virus titer reaches 200-250 ten thousand RID/ml.
Example 6 effect comparison of ST-Sus-Tret-1-4C7 cells with Primary ST-Sus cells in CSFV proliferation application
6.1 ST-Sus-Tret-1-4C7 cells were expressed as 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 2.5X10 6 The cells/ml, CSFV receives the virus according to 2% (volume ratio, seed virus titer is at least 20 ten thousand RID/ml), the virus multiplication of CSFV is carried out by increasing 1 time volume of virus multiplication maintaining liquid (containing trehalose 0.1 um-80 um), the virus multiplication period is 13-15 days, the virus liquid is harvested in 3-5 batches, each batch of virus culture liquid is harvested except the last generation, and fresh virus multiplication maintaining liquid is used for supplementing to the original volume of culture. Specific post-inoculation 2/3 volumes of virus solution were harvested on day 4, 2/3 volumes of virus solution on day 7, 1/2 volumes of virus solution on day 10, 1/2 volumes of virus solution on day 13, and all virus cultures on day 15. The harvested virus can be preserved at 4 to-70 ℃.
The CSFV proliferation potency can reach 200-250 ten thousand RID/ml (different virus potency of different virus harvest generations are different), the virus potency loss is less than 50 ten thousand RID/ml after preservation for one year at 20 ℃, and the virus potency loss is less than 20 ten thousand RID/ml after preservation for one year at 70 ℃.
6.2 the original ST-Sus cells were grown at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 2.5X10 6 CSFV is inoculated with 2% virus (volume ratio, seed virus titer is at least 20 thousand RID/ml), virus proliferation of CSFV is carried out by adding 1 time volume of virus proliferation maintaining solution (containing trehalose 0.1-80 um), virus proliferation period is 13-15 days, virus solution is harvested in 3-5 batches, each batch of virus culture solution is harvested except the last generation, and fresh virus proliferation is maintainedThe liquid is complemented to the original volume of the culture. Specific post-inoculation 2/3 volumes of virus solution were harvested on day 4, 2/3 volumes of virus solution on day 7, 1/2 volumes of virus solution on day 10, 1/2 volumes of virus solution on day 13, and all virus cultures on day 15.
The original ST-Sus cell proliferation potency is 50-100 ten thousand RID/ml (the virus potency of different virus collection generations are different), the virus potency loss is greater than 100 ten thousand RID/ml after one-year preservation at 20 ℃, and the virus potency loss is greater than 50 ten thousand RID/ml after one-year preservation at 70 ℃.
EXAMPLE 7 use of single cell suspension grown ST-Sus-Tret-1-3D3 cells in proliferation of PRV
ST-Sus-Tret-1-3D3 cells were grown at 1X 10 6 Single cell suspension culture is carried out by taking cells/ml as initial culture density, and the density of living cells reaches 3.0x10 6 The cells/ml and PRV are inoculated according to MOI=0.05, the virus proliferation period is 48-66 hours, and the virus titer reaches 9.0-9.5 lgTCID 50 /ml。
Example 8 comparison of the effects of ST-Sus-Tret-1-3D3 cells with Primary ST-Sus cells in PRV proliferation applications
8.1 ST-Sus-Tret-1-3D3 cells were expressed as 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 3.0X10 6 PRV was inoculated at a rate of mol=0.05, and PRV virus was propagated by increasing the volume of virus growth maintaining solution (containing trehalose 0.1 to 80 um) by 1 time, with a period of 48 to 66 hours. The living cells of ST-Sus-Tret-1-3D3 grown by single cell suspension are reduced to 0.1-1.0X10 6 Virus harvest can be performed at cells/ml. The harvested virus can be preserved at 4-70 ℃ for 1 year.
PRV proliferation potency can reach 9.0-9.5 lgTCID 50 Per ml, less than 0.5lgTCID after 3 months of storage at 4 DEG C 50 Per ml, the loss of viral potency after one year of storage at-20 ℃ is less than 1.0lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is less than 0.2lgTCID 50 /ml。
9.2 willPrimitive ST-Sus cells were grown at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 3.0X10 6 PRV was inoculated at a rate of mol=0.05, and PRV virus was propagated by increasing the volume of virus growth maintaining solution (containing trehalose 0.1 to 80 um) by 1 time, with a period of 48 to 66 hours. The living cells of ST-Sus grown in single cell suspension are reduced to 0.1-1.0X10 6 Virus harvest can be performed at cells/ml.
The proliferation potency of the original ST-Sus cells is 8.0-8.5 lgTCID 50 Per ml, the loss of viral titer after 3 months of storage at 4℃is greater than 1.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-20℃is greater than 4.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is greater than 2.0lgTCID 50 /ml。
What has been described above is a specific embodiment of the present invention. It should be noted that modifications and adaptations to the invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Sequence listing
<110> academy of agricultural sciences in Jiangsu province
<120> recombinant ST cells capable of stably expressing TreT, construction method and application thereof
<130> 202010095
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggagctga acaacaagga ggacagcccg cggcacacag ttccttttgt gcgccaaatc 60
acagaagatg ggaaggcgaa gctggaaatt tatcggccga ctaccaaccc catttatatc 120
tacacccaga ttctggcggc catcgctgtg agcatggggt ctatggtggt gggctttgcc 180
tctgcctaca catctcctgc cctggtatcc atgcagaaca ccacaattac ttcttttaaa 240
gtgaccgagc aggaggcctc ctgggtggga ggtatcatgc cactggcggg gctggcgggg 300
ggcatcgctg gaggaccatt tattgagtac ctcgggcgta aaaataccat acttgccacg 360
gctgtgccct tcatcgttgc ctggttactt attgctttcg ccaactccat ctggatggtg 420
ctggctggaa gggccctctc aggattctgc gtgggtatag cgtcgctttc gctgccagtg 480
tacttggggg agaccgtgca gcccgaggtc cggggaaccc ttgggctgct acctacagct 540
tttggaaaca tcggaatcct tatctgcttc gtagcaggca aatatgtgaa ctggagtgga 600
ctcgctttta tcgggagcat tttgcccatt cctttcatgg ttctgaccct gctgatccca 660
gagactccac gctggtttgt gacgcgcggg cgagaagaaa gagccagaaa agcactgcag 720
tggctccgtg gcaagaaggc agatgtcgaa cccgaactga aaggtatagt gaagagccac 780
tgtgaagcag agcgccatgc ttcacagaat gccatctttg acctcatgaa gaggagcaat 840
ctgaaaccgc tccttattgc tttaggcttg atgtttttcc agcagctgag cggtattaat 900
gcagtgattt tttatacagt gagtatcttc aaggacgccg gcagtacaat cgatgagaac 960
ttatgcacca taatcgtcgg cgtagtcaat tttggggcca ctttctttgc aactgtgttg 1020
attgacagac tggggaggaa aattctcctc tacatctccg aagtggccat ggtcatcacg 1080
ttgttaactc tgggcacctt cttctactac aagaattcag gaaatgatgt tagtaacatt 1140
ggctggctcc ccctagcatc cttcgtcatc tatgtcattg ggttctcctc tggagttggc 1200
cccatcccct ggttgatgct gggagaaatc ctccctggaa agatccgggg ttctgctgca 1260
agtgtggcga cgggcttcaa ctggacctgt actttcattg tcacaaagac ttttgctgac 1320
atcgtggctg ccattggcaa tcatggcgcc ttctggttct ttggcgttat atgtctgatt 1380
ggcctcttct tcgtaatatt ttttgttcca gaaacacaag gtaaatccct ggaggagata 1440
gagaggaaga tgatgggccg agtcaggaga atgagtagcg tcgcaaacat gaaaccttta 1500
tcattcaata tggattacaa ggacgacgat gacaagtga 1539
Claims (7)
1. A recombinant ST cell (ST-Sus-TreT-1) capable of stably expressing a trehalose transporter (TreT), characterized in that: the cell is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.21010 in the year 11 and 6 of 2020.
2. The recombinant ST cell of claim 1, wherein: the recombinant ST cells contain trehalose transporter coding sequence Tret-1 shown in SEQ ID NO. 1.
3. The method for constructing a recombinant ST cell according to claim 1, comprising the steps of:
(1) Constructing a vector pCMV-Tret-1 containing a trehalose transporter coding sequence;
(2) The transfection complexes were added drop wise to ST cell culture broth, transfected, and the recombinant ST cells stably and continuously grown in resistance were selected using G418 (ST-Sus-Tret-1).
4. A method of construction according to claim 3, wherein: the ST cells are ST-Sus cells grown in single cell suspension.
5. The construction method according to claim 3 or 4, wherein: in the step (2), two rounds of screening are carried out by using G418, wherein the screening concentration of the first round is 500ug/ml, and the screening concentration of the second round is 1000ug/ml.
6. The construction method according to claim 5, wherein: each round used 20 successive rounds of screening.
7. Use of the recombinant ST cell (ST-Sus-Tret-1) of claim 1 for the proliferation of viruses, such as epidemic diarrhea virus (PEDV), proliferation of swine fever virus (CSFV), pseudorabies virus (PRV).
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| US7892789B2 (en) * | 2007-03-05 | 2011-02-22 | Takahiro Kikawada | Method of increasing cell permeability to trehalose by recombinantly producing a trehalose transporter |
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