CN114958757A - Recombinant ST cell capable of stably expressing TreT and construction method and application thereof - Google Patents
Recombinant ST cell capable of stably expressing TreT and construction method and application thereof Download PDFInfo
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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 transporter, and a construction method and application thereof. The recombinant ST cell capable of stably expressing the trehalose transporter is obtained by a bioengineering method, and the ST cell with trehalose accumulation capacity is obtained by adding trehalose into a culture environment. Experiments show that the recombinant ST cell can be used for proliferating the virus, so that the virus survival rate is remarkably improved at 37 ℃, and the cell can remarkably maintain the virus survival rate under 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 transporter, and a construction method and application thereof.
Background
Trehalose is a non-reducing disaccharide made from two molecules of glucopyranose linked by alpha, alpha-1, 1 glycosidic bonds. Has stable chemical properties. The trehalose can provide effective protection for biomacromolecules in extreme environments such as high temperature, low temperature, high osmotic pressure, dryness and the like.
Chironomus aquatica (Polypedilum vanderplaki) larvae can accumulate trehalose in large quantities in extreme environments such as dry or salt stress, and thus regain activity after leaving the extreme environment. Accordingly, aquatic chironomids are also known as "dehydrated organisms". The reason for this is that aquatic chironomid larvae can express Trehalose transporter (TreT) in extreme environments, thereby transporting Trehalose to the inside of the body and realizing the accumulation of Trehalose.
ST cells, namely porcine testicular cells (swine testis), are widely used in the proliferation process of enveloped viruses.
The ST cell with the capacity of synthesizing or transferring the trehalose is obtained by a biotechnology means so as to improve the survival rate of the progeny virus in a low-temperature freezing environment, which has positive effects and important significance for products taking the virus as a basic raw material.
Therefore, it is a research focus of those skilled in the art to find an ST cell capable of synthesizing or transporting trehalose. However, there is no report on the synthesis and transport of trehalose in mammals, and therefore, this also becomes a difficult point for those skilled in the art to study.
Disclosure of Invention
Aiming at the report that trehalose synthesis and transportation are not related in mammals at present, the invention discloses a recombinant ST cell capable of stably expressing trehalose transporter, a construction method of the cell and application of the cell in virus proliferation, so that the trehalose in a culture environment can be transported and accumulated into the cell by an animal cell.
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 for short) at 11 months and 6 days in 2020, with the preservation number of CGMCC NO.21010 and the preservation address of China institute of sciences, institute of microbiology, No. 3 of Ministry of China, North Jing city, morning area, North Chen Xilu No. 1.
The recombinant ST cell contains a trehalose transport protein coding sequence Tret-1 shown as SEQ ID:1, wherein the trehalose transport protein coding sequence Tret-1 is a recombinant nucleotide sequence optimized according to the pig codon preference.
Meanwhile, the invention also discloses a construction method of the recombinant ST cell, which comprises the following steps:
(1) constructing a carrier pCMV-Tret-1 containing a trehalose transporter coding sequence;
(2) the transfection complex was added dropwise to the ST cell culture medium, transfected, and G418 was used to screen for stable and sustained growth of recombinant ST-Sus-Tret-1 cells in resistance.
Wherein the ST cells are single cell suspension grown ST-Sus cells.
Preferably, two rounds of screening are carried out in step (2) using G418, the concentration of the first round of screening is 500ug/ml, and the concentration of the second round of screening is 1000 ug/ml. Preferably, 20 consecutive screenings are used per round.
Further, the invention also discloses the application of the recombinant ST cell (ST-Sus-Tret-1) in virus propagation.
Specifically, in the invention, single cell clone screening is further carried out on the recombinant ST cell (ST-Sus-Tret-1) by the target virus to be proliferated, so as to obtain the single cell clone aiming at the target virus, thereby being used for the proliferation of the virus.
In a preferred technical scheme, the recombinant ST cell ST-Sus-Tret-1 is applied to the proliferation of Porcine Epidemic Diarrhea Virus (PEDV).
Further, the invention also discloses a single-cell clone ST-Sus-Tret-1-9E2 for proliferating the Porcine Epidemic Diarrhea Virus (PEDV).
In a preferred embodiment, the recombinant ST cell (ST-Sus-Tret-1) is used for the propagation of Classical Swine Fever Virus (CSFV).
Furthermore, the invention also discloses a single-cell clone ST-Sus-Tret-1-4C7 for proliferating the Classical Swine Fever Virus (CSFV).
In a preferred embodiment, the recombinant ST cell (ST-Sus-Tret-1) is used for the proliferation of pseudorabies virus (PRV).
Further, the invention also discloses a single-cell clone ST-Sus-Tret-1-3D3 for propagating the pseudorabies virus (PRV).
The invention obtains the recombinant ST cell capable of stably expressing trehalose transporter by a bioengineering method, and further obtains the ST cell with trehalose accumulation capacity by adding trehalose into a culture environment. Experiments show that the recombinant ST cell can obviously improve the survival rate of the virus at 37 ℃ and shows an obvious maintenance effect on the survival rate of the virus aiming at different freezing conditions by proliferating the virus through the cell.
Drawings
FIG. 1 is an enzyme cutting electrophoresis diagram of a Tret-1 recombinant expression vector;
wherein M: the DNA marker, 1: pCMV-Tret-1 is subjected to double enzyme digestion treatment by NheI and NotI.
FIG. 2 shows the recombinant expression assay of Tret-1 from a cell clone that restores adherent growth after FACS sorting of ST-Sus-Tret-1 cells.
Detailed Description
In order that the invention may be better understood, we now provide further explanation of the invention with reference to specific examples.
Example 1 construction of ST-Sus-Tret-1 cells
1.1 vector construction
Optimizing the nucleotide sequence codon of trehalose transport protein (Tret) according to the pig codon preference, wherein the optimized recombinant nucleotide sequence Tret-1 is shown as SEQ ID NO. 1. This sequence was cloned into the pUC57 vector to obtain the pUC-Tret-1 vector.
And (2) amplifying the fragment from a pUC-Tret-1 plasmid containing a Tret-1 coding sequence by using an upstream primer and a downstream primer with NheI and NotI enzyme cutting sites, cloning the fragment into an equivalent double-enzyme-cut eukaryotic expression vector after double enzyme cutting, and constructing the eukaryotic expression vector pCMV-Tret-1 of the Tret-1 for transfection operation.
Specifically, the primers p1 and p2 used in this example were as follows:
p1：GCCGGTACCATGGAGCTGAACAACAAGGAGGAC (NheI restriction site in the underlined position)
p2：GCCGCGGCCGCTCACTTGTCATCGTCGTCCTTG (NotI cleavage site in the underlined)
And (3) amplifying a coding sequence of the Tret-1 by using pUC-Tret-1 as a template through PCR reaction, and adding a flag tag at the C terminal. The target fragment amplified by PCR and the pCMV plasmid are treated by NheI and NotI double enzyme digestion to construct a recombinant eukaryotic expression vector pCMV-Tret-1, and the enzyme digestion identification is shown in figure 1. The plasmid for transfection with higher quality is obtained by extraction with a plasmid large-extraction kit of Qiagen company.
1.2 Generation of ST-Sus-Tret-1 cells
The pCMV-Tret-1 is transfected into the ST-Sus cells with single cell suspension growth, and the G418 drugs are used for two rounds of screening to obtain the recombinant ST cells which have resistance to the G418 drugs and can stably express the Tret-1 protein.
Specifically, ST-Sus cells were first cultured at an initial cell density of 5X 10 5 cells/ml were inoculated in 50ml Falcon tubes at 15ml, 180rpm, 37 ℃ with 5% CO 2 After overnight incubation, the cells were used for transfection.
The medium was changed to Opti-MEM medium before transfection and incubated in an incubator for 10 minutes.
The transfection complex was then configured according to the instructions for the 25kDa branched PEI transfection reagent and incubated for 5 minutes at room temperature. After the incubation is completed, the transfection complex is added dropwise to the ST-Sus cells, and the cells are placed in an incubator for 6-8 hours with intermittent agitation.
After transfection was completed, the culture supernatant containing the remaining transfection complex was removed and replaced with normal medium for suspension culture.
The ST-Sus cells 24-72 hours after transfection were re-inoculated into a 50ml Falcon culture tube at a ratio of 1: 3-1: 5 at 180rpm, 37 ℃ and 5% CO 2 And G418 culture solution containing 500ug/ml for recombinant cell screening. And G418 continuous screening is carried out according to the frequency of changing the liquid once in 3 days, and the first screening time is about 2-3 weeks (20 continuous generations). Recombinant ST-Sus-Tret-1 cells capable of sustained proliferation under the pressure of G418 selection were selected for continued culture. In the second round of screening, the working concentration of G418 screening is increased to 1000ug/ml, and pressurized screening is continued for 20 generations according to the first round of screening method, so as to finally obtain the recombinant ST-Sus-Tret-1 cells which can stably and continuously grow in the G418 screening resistance.
EXAMPLE 2 preparation of the Single cell clone ST-Sus-Tret-1-9E2, the Single cell clone ST-Sus-Tret-1-4C7 and the Single cell clone ST-Sus-Tret-1-3D3
Screening the resistant stable and continuous-growth recombinant ST-Sus-Tret-1 cells obtained in example 1, sorting the cells into 96-well plates by a flow cytometer, forming single cell clones in each well, forming 173 cell clones with effective adherent growth after the cells recover adherent growth, making a cell clone replate, and detecting the recombinant expression of the Tret-1 of the cell clones as shown in figure 2.
Aiming at the proliferation of PEDV, CSFV and PRV, the single-cell clones are screened by adopting PEDV, CSFV and PRV, the cell clone which has the best proliferation to the three viruses is screened from 173 cell clones, and the cell clones are named respectively to obtain the single-cell clone ST-Sus-Tret-1-9E2 aiming at the efficient proliferation of PEDV, the single-cell clone ST-Sus-Tret-1-4C7 aiming at the efficient proliferation of CSFV and the single-cell clone ST-Sus-Tret-1-3D3 aiming at the efficient proliferation of PRV.
Example 3 application of Single cell suspension grown ST-Sus-Tret-1-9E2 cells to proliferation of PEDV
ST-Sus-Tret-1-9E2 cells at 1X 10 6 cell/ml is used as initial culture density to carry out single cell suspension culture under the culture condition of 180rpm and 37℃，5％CO 2 And a serum-free suspension growth medium for ST-Sus cells.
When the living cell density reaches 4.5 multiplied by 10 6 cells/ml, PEDV is inoculated according to the MOI of 0.1, the virus propagation period is 24-36 hours, and the virus titer reaches 8.75-9.0 lgTCID 50 /ml。
Example 4 comparison of the Effect of ST-Sus-Tret-1-9E2 cells with Primary ST-Sus cells in PEDV proliferation applications
4.1 ST-Sus-Tret-1-9E2 cells at 1X 10 6 cell/ml is used as initial culture density to perform single cell suspension culture under the conditions of 180rpm, 37 ℃ and 5% CO 2 And a serum-free suspension growth medium for ST-Sus cells. When the living cell density reaches 10 multiplied by 10 6 cells/ml, PEDV virus proliferation is carried out by adding virus proliferation maintenance liquid (containing trehalose of 0.1-80 um) with the volume increased by 1 time according to the MOI of 0.3 for virus inoculation, and the proliferation cycle is 24-36 hours.
The viable cells of the single-cell suspension-grown ST-Sus-Tret-1-9E2 are reduced to 0.1-1.2X 10 6 The cells/ml can be used for virus harvest.
The harvested viruses can be preserved at 4-70 ℃, and the preservation time can reach 1 year. The proliferation potency of PEDV can reach 8.75-9.0 lgTCID 50 Viral titer loss after one year storage at-20 ℃ of less than 0.5lgTCID 50 Per ml, a loss of viral titer after one year storage at-70 ℃ of less than 0.2lgTCID 50 /ml。
4.2 Primary ST-Sus cells at 1X 10 6 cells/ml as initial culture density, and culturing at 180rpm, 37 deg.C and 5% CO 2 And a serum-free suspension growth medium for ST-Sus cells. When the living cell density reaches 10 multiplied by 10 6 cells/ml, PEDV was inoculated according to MOI of 0.3, virus growth maintenance solution (containing trehalose of 0.1-80 um) with 1-fold volume was added to proliferate PEDV virus, and the proliferation cycle was 24-36 hours.
The viable cells of the single-cell suspension-grown ST-Sus are reduced to 0.1-1.2 multiplied by 10 6 The cells/ml can be used for virus harvest.
The original ST-Sus cell proliferation titer is 7.33-7.77 lgTCID 50 /ml，-20℃The loss of virus titer after one year of storage is more than 2.5lgTCID 50 Viral titer loss after one year storage at-70 ℃ of more than 1.5lgTCID 50 /ml。
Example 5 application of Single cell suspension grown ST-Sus-Tret-1-4C7 cells to the proliferation of CSFV
ST-Sus-Tret-1-4C7 cells at 1X 10 6 cell/ml is used as initial culture density to carry out single cell suspension culture until the living cell density reaches 2.5 multiplied by 10 6 cells/ml, inoculating CSFV according to 2% (volume ratio), collecting virus liquid in 3-5 batches with virus multiplication period of 13-15 days, and obtaining virus titer of 200-250 ten thousand RID/ml.
Example 6 comparison of the Effect of ST-Sus-Tret-1-4C7 cells with Primary ST-Sus cells in CSFV proliferation applications
6.1 ST-Sus-Tret-1-4C7 cells at 1X 10 6 cell/ml is used as initial culture density to perform single cell suspension culture under the conditions of 180rpm, 37 ℃ and 5% CO 2 And a serum-free suspension growth medium for ST-Sus cells. When the living cell density reaches 2.5 multiplied by 10 6 cell/ml, adding virus multiplication maintenance liquid (containing trehalose of 0.1-80 um) with the volume 1 time that of CSFV according to 2% virus inoculation (volume ratio, seed toxicity titer is at least 20 ten thousand RID/ml) to carry out virus multiplication of CSFV, wherein the virus multiplication cycle is 13-15 days, harvesting virus liquid in 3-5 batches, harvesting one batch of virus culture liquid except the last batch, and supplementing fresh virus multiplication maintenance liquid to the original volume of culture. Specifically, 2/3 volumes of virus solution were harvested on day 4, 2/3 volumes of virus solution were harvested on day 7, 1/2 volumes of virus solution were harvested on day 10, 1/2 volumes of virus solution were harvested on day 13, and all virus cultures were harvested on day 15 after inoculation. The harvested virus can be preserved at 4-70 ℃.
The CSFV multiplication titer can reach 200-250 ten thousand RID/ml (the virus titers of different virus generations are different), the virus titer loss is less than 50 ten thousand RID/ml after being preserved for one year at 20 ℃, and the virus titer loss is less than 20 ten thousand RID/ml after being preserved for one year at 70 ℃.
6.2 Primary ST-Sus cells at 1X 10 6 cell/ml is used as initial culture density to perform single cell suspension culture under the conditions of 180rpm, 37 ℃ and 5% CO 2 And a serum-free suspension growth medium for ST-Sus cells. When the living cell density reaches 2.5 multiplied by 10 6 cell/ml, adding 1 time volume of virus multiplication maintaining liquid (containing trehalose of 0.1-80 um) into CSFV according to 2% virus inoculation (volume ratio, seed virus titer is at least 20 ten thousand RID/ml) to carry out virus multiplication of CSFV, wherein the virus multiplication period is 13-15 days, harvesting virus liquid in 3-5 batches, harvesting virus culture liquid in each batch except the last generation, and supplementing fresh virus multiplication maintaining liquid to the original volume of culture. Specifically, 2/3 volumes of virus solution were harvested on day 4, 2/3 volumes of virus solution were harvested on day 7, 1/2 volumes of virus solution were harvested on day 10, 1/2 volumes of virus solution were harvested on day 13, and all virus cultures were harvested on day 15 after inoculation.
The original ST-Sus cell proliferation titer is 50-100 ten thousand RID/ml (virus titers of different virus generations are different), the virus titer loss is more than 100 ten thousand RID/ml after being preserved for one year at 20 ℃, and the virus titer loss is more than 50 ten thousand RID/ml after being preserved for one year at 70 ℃.
Example 7 application of Single cell suspension grown ST-Sus-Tret-1-3D3 cells to proliferation of PRV
ST-Sus-Tret-1-3D3 cells at 1X 10 6 cell/ml is used as initial culture density to carry out single cell suspension culture until the living cell density reaches 3.0 × 10 6 cells/ml, PRV according to MOI 0.05 virus inoculation, virus propagation cycle is 48-66 hours, virus titer reaches 9.0-9.5 lgTCID 50 /ml。
Example 8 comparison of the Effect of ST-Sus-Tret-1-3D3 cells with Primary ST-Sus cells in PRV proliferation applications
8.1 ST-Sus-Tret-1-3D3 cells at 1X 10 6 cell/ml is used as initial culture density to perform single cell suspension culture under the conditions of 180rpm, 37 ℃ and 5% CO 2 And a serum-free suspension growth medium for ST-Sus cells. When the living cell density reaches 3.0X 10 6 cells/ml, PRV increases 1 time volume of virus multiplication maintenance liquid (containing trehalose 0.1-80 um) according to MOI 0.05 virus inoculation, and PRV virus multiplication cycle is 48-66 hours. The living cells of the single-cell suspension-grown ST-Sus-Tret-1-3D3 are reduced to 0.1-1.0X 10 6 cells/ml is justAnd (5) harvesting the virus. The harvested virus can be preserved at 4-70 ℃ for 1 year.
The PRV proliferation potency can reach 9.0-9.5 lgTCID 50 Per ml, a loss of viral titer after 3 months storage at 4 ℃ of less than 0.5lgTCID 50 Viral titer loss after one year storage at-20 ℃ of less than 1.0lgTCID 50 Viral titer loss after one year storage at-70 ℃ of less than 0.2lgTCID 50 /ml。
9.2 Primary ST-Sus cells at 1X 10 6 cell/ml is used as initial culture density to perform single cell suspension culture under the conditions of 180rpm, 37 ℃ and 5% CO 2 And a serum-free suspension growth medium for ST-Sus cells. When the living cell density reaches 3.0 multiplied by 10 6 cells/ml, PRV increases 1 time volume of virus multiplication maintenance liquid (containing trehalose 0.1-80 um) according to MOI 0.05 virus inoculation, and PRV virus multiplication cycle is 48-66 hours. The viable cells of the single-cell suspension-grown ST-Sus are reduced to 0.1-1.0 x 10 6 The cells/ml can be used for virus harvest.
The original ST-Sus cell proliferation titer is 8.0-8.5 lgTCID 50 Per ml, a loss of viral titer after 3 months storage at 4 ℃ of more than 1.5lgTCID 50 Viral titer loss after one year storage at-20 ℃ of greater than 4.5lgTCID 50 Viral titer loss after one year storage at-70 ℃ of more than 2.0lgTCID 50 /ml。
What has been described above is a specific embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.
Sequence listing
<110> agricultural science and academy of Jiangsu province
<120> recombinant ST cell capable of stably expressing TreT and 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 (10)
1. Recombinant ST cells (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) at 11/6/2020 with the preservation number of CGMCC NO. 21010.
2. The recombinant ST cell of claim 1, wherein: the recombinant ST cell contains a trehalose transport protein coding sequence Tret-1 shown in SEQ ID: 1.
3. The method of constructing a recombinant ST cell of claim 1, comprising the steps of:
(1) constructing a vector pCMV-Tret-1 containing a trehalose transporter coding sequence;
(2) the transfection complex was added dropwise to the culture of ST cells, transfected and G418 was used to select stable and sustained growing recombinant ST-Sus-Tret-1 cells in resistance.
4. The construction method according to claim 3, characterized in that: the ST cells are single cell suspension grown ST-Sus cells.
5. The construction method according to claim 3 or 4, characterized in that: in the step (2), G418 is used for two-round screening, the screening concentration of the first round is 500ug/ml, and the screening concentration of the second round is 1000 ug/ml; preferably, 20 consecutive screenings are used per round.
6. Use of the recombinant ST cell of claim 1 (ST-Sus-Tret-1) for propagating a virus.
7. Use according to claim 6, characterized in that: the virus is epidemic diarrhea virus (PEDV), proliferating Classical Swine Fever Virus (CSFV) and pseudorabies virus (PRV).
8. A single cell clone ST-Sus-Tret-1-9E2 for the propagation of Porcine Epidemic Diarrhea Virus (PEDV).
9. A single cell clone ST-Sus-Tret-1-4C7 for the propagation of Classical Swine Fever Virus (CSFV).
10. A single cell clone ST-Sus-Tret-1-3D3 for the proliferation of pseudorabies virus (PRV).
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