CN109913423A - A kind of the recombination Vero cell line and application of stable expression pig Delta coronavirus N protein - Google Patents
A kind of the recombination Vero cell line and application of stable expression pig Delta coronavirus N protein Download PDFInfo
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Abstract
The invention belongs to field of biological technology detection, and in particular to a kind of recombinant cell lines and its application of stable expression pig Delta coronavirus N protein.The cell line contains pig Delta coronavirus N protein gene and resistance screening gene.The recombinant cell lines with puromycin-resistant for stablizing expression PDCoV-N albumen that the present invention obtains, it can be used for detecting PDCoV specific antibody, for the swinery without vaccine immunity, monitors specific antibody positive reaction i.e. and can determine that the swinery has occurred and that wild virus infection.
Description
Technical field
The invention belongs to field of biological technology detection, and in particular to a kind of stable expression pig Delta coronavirus N protein
Recombinant cell lines and its application.
Background technique
Pig Delta coronavirus (Porcine deltacoronavirus, PDCoV) is a kind of newfound coronal disease
Poison can cause pig, especially newborn piglet diarrhea, and the clinical manifestation and known transmissible gastroenteritis of swine that cause and pig are popular
Property diarrhea it is closely similar, be easy to obscure, can lead to higher morbidity and mortality after infection, be in recent years it is multiple country and
Cause the one of the major reasons of newborn piglet death in area.PDCoV most had found (Woo by Hong Kong scholar earlier than 2012 for the first time
PC,et al.Discovery of seven novel Mammalian and avian coronaviruses in the
genus deltacoronavirus supports bat coronaviruses as the gene source of
alphacoronavirus and betacoronavirus and avian coronaviruses as the gene
source of gammacoronavirus and deltacoronavirus.Journal of virology,2012,86
(7): 3995-4008.), extensive prevalence (Li G, et al.Full-Length Genome occurs for the first time in the U.S. within 2014
Sequence of Porcine Deltacoronavirus Strain USA/IA/2014/8734.Genome
Announcements, 2014,2 (2): e00278-14.), then in South Korea, the ground such as China are detected (Lee S, et in succession
al.Complete Genome Characterization of Korean Porcine Deltacoronavirus Strain
KOR/KNU14-04/2014.Genome announcements, 2014,2 (6): e01191-14.；Chen F,et
al.2015.Full-length genome characterization of Chinese porcine
deltacoronavirus strain CH/SXD1/2015.Gen ome Announcements,3(5):e01284-15；),
Show the potential trend of Global prevalence.There is no effective antiviral therapy drug and vaccine for the infection of the virus at present,
Effective means are also lacked to the monitoring of its epidemic situation.Nucleocapsid protein (N) is one of most important structural proteins of PDCoV, virus sense
The specific antibody for the albumen can be generated after dye rapidly, therefore the recombinant cell lines for establishing expression N protein can be used for pig
Group carries out PDCoV and infects antibody detection.
Summary of the invention
The purpose of the invention is to provide a kind of protein stabilized expression cell system of PDCoVN and its applications.
The principle of the present invention and most crucial key technology are scientifically and rationally to construct the slow disease of insertion PDCoVN gene
It is big to be transformed into Stbl2 competent cell later by malicious system expression plasmid pLVX-EF1 α-IRES-Puro-PDCoV-N for the plasmid
Amount amplification, the packaging of slow virus is carried out with 2 helper plasmid psPAX2 and pMD2.G cotransfection 293T cells, with the slow disease of collection
Malicious vero cells infection, if successfully infecting, slow virus carrier can be by the puromycin on the PDCoV-N gene and carrier of carrying
Resistant gene is integrated into Vero cellular genome together, pressurizes and screens through certain density puromycin, and passes through clone purification
The recombinant cell lines with puromycin-resistant for stablizing expression PDCoV-N albumen of acquisition can be used for detecting PDCoV specificity
Antibody monitors specific antibody positive reaction i.e. and can determine that the swinery has occurred and that open country for the swinery without vaccine immunity
Poison infection.
Realizing the technical solution of the object of the invention is:
A kind of recombination Vero cell line of stable expression pig Delta coronavirus N protein, which is characterized in that the cell line
Contain pig Delta coronavirus N protein gene and resistance screening gene.
Specifically, which contains pig Delta coronavirus N protein gene and puromycin resistance gene, is Africa
Green monkey kidney cell Vero/PDCoV-N, its deposit number are CCTCC NO:C2018255.
The invention also discloses the recombination Vero cell lines of the stable expression pig Delta coronavirus N protein to prepare
Detect the application in pig Delta coronavirus reagent.
A kind of stable expression PDCoVN Protein reconstitution Vero cell line of the present invention, can be obtained by following steps
It arrives:
(1) PCR amplification aim sequence, according to the PDCoVCHN/Tianjin/2016 of this experimental determination strain N gene order
(GenBank accession number: KY065120) removes end terminator codon TAG, uses primer-design software Primer Premier
5 pairs of remaining base sequence design primers carry out PCR amplification.For convenience of clone and subsequent identification, genetic fragment both ends add respectively
Upper BamH I and Xho I restriction enzyme enzyme recognition site, while Flag sequence label is added in 3 end ' of gene.Design
PCR primer are as follows:
PDCOV-N-F (SEQ ID NO.1): 5'-GAA CTC GAG ATC ATG GCT GCA CCA GTA GTC-3'
PDCOV-N-R (SEQ ID NO.2): 5'-GTT GGA TCC CTA CTT GTC GTC ATC GTC TTT
GTAGTC CGC TGC TGA TTCTT-3'
(2) PCR product obtained in step (1) building of recombinant slow virus expression plasmid: is subjected to Ago-Gel electricity
Swimming identification identifies correct progress gel extraction and the target fragment directed cloning of recycling is entered pLVX-EF1 α-IRES-Puro carrying
Between body corresponding site, obtains recombinant slow virus expression vector pLVX-EF1 α-IRES-Puro-PDCoV-N (see Fig. 1), will weigh
Group expression vector is further transformed into Stbl2 competent cell, and the recombinant bacterium of acquisition is named as Stbl2/pLVX-EF1 α-IRES-
Puro-PDCoV-N。
(3) packaging of slow virus: by the recombinant bacterium Stbl2/pLVX-EF1 α-IRES-Puro-PDCoV-N built with 1:
100 ratio is inoculated in the LB liquid medium of addition ampicillin, is used after the violent 12~16h of shake culture of 250r/min
It goes endotoxin to extract plasmid kit and extracts pLVX-EF1 α-IRES-Puro-PDCoV-N plasmid, by the pLVX-EF1 α-of extraction
IRES-Puro-PDCoV-N slow virus expression plasmid and 2 helper plasmids pMD2.G and psPAX2 are used3000
Transfection reagent cotransfection 293T cell carries out viral packaging (15 μ g of plasmid gross mass, the ratio of plasmid are followed successively by 7:2:1), 48h
Supernatant is collected after~72h, interval 48h~72h is regathered, collected 3 times altogether, the slow virus supernatant 1000r/min centrifugation of collection
10min discards bottom cell precipitation, is stored in -70 DEG C.
(4) determination of puromycin screening concentration: being inoculated with Vero cell, totally 11 hole in 24 orifice plates, overnight incubation, cell
It grows to floor space 80%~90%, successively changes the complete medium (10%FBS-DMEM) that joined puromycin to every hole,
The concentration gradient of 0,1,2,3,4,5,6,7,8,9,10 μ g/ml is set, and every 2 days observation cell survival conditions, (cell was hiked up i.e.
For death), every the culture medium of 2 days replacement 0.75mL puromycins containing respective concentration.Screening keeps cell all dead in 4~7 days
The minimum puromycin concentration died, as screening concentration.4th day, the Vero cell in the hole containing 4 μ g/ml puromycins was dead
Complete (see Fig. 3), determine the puromycin complete medium containing 4 μ g/ml be after screening and culturing medium.
(5) slow-virus infection Vero cell and puromycin screening: suitable Vero cell is inoculated in 12 porocyte plates
2 holes, 1 hole infects slow virus, and another 1 hole is as control, when cell grows into 80%~90% floor space of covering, abandons
Wherein 1 hole culture medium is removed, is cleaned 2~3 times with sterile PBS (pH7.2).Aspiration step (3) collect slow virus 0.5mL and
0.5mL DMEM is uniformly mixed and is inoculated on cell, discards slow virus DMEM mixed liquor addition complete medium after infecting 6~8h,
After 48h, change the culture medium in 2 holes into puromycin screening and culturing medium, every 2 days replacement primary screening culture mediums, until pair
According to hole inner cell all it is dead it is complete will connect malicious hole inner cell and all produce to another 1 12 empty holes, carried out 2 times with screening and culturing medium
Screening, cell discards 4/5 cell after covering with continue screening passage, so expand culture after 4~5 generations of screening, obtain cell line,
It is named as Vero/PDCoV-N, China typical culture collection center is preserved on December 10th, 2018, address China is military
Chinese Wuhan University, deposit number CCTCC NO.C2018255.
The beneficial effects of the present invention are embodied in: N protein be after coronavirus infection host cell in virus replication
The early protein of generation and the main structural proteins of coronavirus, earliest may be used in coronavirus infection postoperative infection animal body
The antibody of detection is the antibody for N protein, therefore detects N protein antibody and can be used for the early diagnosis of virus infection.This hair
The cell line Vero/PDCoV-N energy specificity based on N protein of bright acquisition detects PDCoV antibody, and popular with anti-pig
Diarrhea virus positive serum (PEDV), anti-transmissible gastroenteritis virus-positive serum (TGEV), anti-rotavirus positive serum
(RoV) and pig negative serum does not react, therefore the cell line has good specificity.Due to not yet developing both at home and abroad at present
Out prevention PDCoV infection vaccine, as long as therefore swinery detect N protein antibody positive, so that it may be determined as PDCoV infection.This
Invention carries out PDCoV pathogeny detection relative to by nucleic acid detection method, and the present invention is more applicable for PDCoV infection epidemic disease
Learn the high flux examination in investigation for a large amount of samples.
Detailed description of the invention
Fig. 1 is the recombinant expression carrier pLVX-EF1 α-IRES-Puro-PDCoV-N structural schematic diagram that the present invention constructs
Fig. 2 is that recombinant expression carrier pLVX-EF1 α-IRES-Puro-PDCoV-N is identified through BamH I and Xho I double digestion
Electrophoretogram (M:DNA Marker；Swimming lane 1: it can produce after recombinant expression carrier pLVX-EF1 α-IRES-Puro-PDCoV-N digestion
The carrier ribbon of 8.8kb size and the purpose band of 1074bp size；Swimming lane 2:pLVX-EF1 α-IRES-Puro empty carrier digestion
The carrier ribbon of 8.8kb size is only observed afterwards)
Fig. 3 is cellular morphology schematic diagram (the figure A: the Vero cell shape after screening and culturing medium culture 4 days that puromycin kills
State；Scheme B: the Vero cellular morphology of normal growth).
Fig. 4 is PCR qualification figure (the M:DNA Marker of cell line Vero/PDCoV-N；Swimming lane 1:Vero cell total rna
RT-PCR product；Swimming lane 2: the total serum IgE RT-PCR product of recombinant cell lines Vero/PDCoV-N).
Fig. 5 is that recombinant cell lines Vero/PDCoV-N uses the monoclonal antibody for Flag label to carry out as primary antibody
The result figure (M: albumen Marker of Western-blot identification；Swimming lane 1: recombinant cell lines Vero/PDCoV-N albumen sample；Swimming lane
2:Vero cell protein sample).
Fig. 6, which is recombinant cell lines Vero/PDCoV-N, carries out Western- with the monoclonal antibody for PDCoV-N albumen
The result figure (M: albumen Marker of blot identification；Swimming lane 1: cell line Vero/PDCoV-N albumen sample；Swimming lane 2:Vero cell egg
White sample).
Fig. 7 be cell line Vero/PDCoV-N immunohistochemical staining test result (A: primary antibody be pig negative serum；B: one
Resist and infect PDCoV positive serum for pig).
Fig. 8 be cell line Vero/PDCoV-N indirect immunofluorescence assay result (A: primary antibody be pig negative serum；B: one
Resist and infect PDCoV positive serum for pig).
Cell line Vero/PDCoV-N of the invention is preserved in China typical culture collection on December 10th, 2018
Center, address Wuhan, China Wuhan University, deposit number CCTCC NO:C2018255, classification naming are as follows: African green monkey kidney
Cell Vero/PDCoV-N.
Specific embodiment
It will be specifically described, but be not to be construed as by the way that attached drawing and specific embodiment party example are further to the present invention below
Limiting the scope of the present invention.
The building of 1 recombinant expression carrier pLVX-EF1 α-IRES-Puro-PDCoV-N of embodiment
PCR amplification aim sequence, according to the PDCoVCHN/Tianjin/2016 of this experimental determination strain N gene order
(Gen Bank accession number: KY065120), removes end terminator codon TAG, uses primer-design software Primer
5 design primer of Premier carries out PCR amplification.For convenience of clone, genetic fragment both ends add BamH I and Xho I limitation respectively
Property endonuclease recognized site；For convenience of subsequent identification, Flag sequence label is added in 3 end ' of gene.The PCR primer of design
Are as follows:
PDCOV-N-F (SEQ ID NO.1): 5'-GAA CTC GAG ATC ATG GCT GCA CCA GTA GTC-3'
PDCOV-N-R (SEQ ID NO.2): 5'-GTT GGA TCC CTA CTT GTC GTC ATC GTC TTT
GTAGTC CGC TGC TGA TTCTT-3'
The Flag sequence label of addition are as follows: 5'-CTT GTC GTC ATC GTC TTT GTA GTC-3'(SEQ ID
NO.3)
The building of recombinant slow virus expression plasmid: PCR product obtained in step (1) is subjected to agarose gel electrophoresis mirror
It is fixed, it identifies correct progress gel extraction and the target fragment directed cloning of recycling is entered into pLVX-EF1 α-IRES-Puro carrier phase
It answers between site, obtains recombinant slow virus expression vector pLVX-EF1 α-IRES-Puro-PDCoV-N (see Fig. 1), recombinant vector
After BamH I and Xho I double digestion through agarose gel electrophoresis identification can be observed 8.8kb size carrier ribbon and
The purpose band of 1074bp size, and the load of 8.8kb size is only observed after pLVX-EF1 α-IRES-Puro empty carrier double digestion
Body band (see Fig. 2).Recombinant expression plasmid is further transformed into Stbl2 competent cell, the recombinant bacterium of acquisition is named as
Stbl2/pLVX-EF1α-IRES-Puro-PDCoV-N。
The foundation of 2 recombinant cell lines Vero/PDCoV-N of embodiment
The recombinant cell lines Vero/PDCoV-N of detection PDCoV antibody of the present invention, can be obtained by following step:
The packaging of slow virus: by the recombinant bacterium Stbl2/pLVX-EF1 α-IRES-Puro-PDCoV-N built with 1:100
Ratio be inoculated in addition ampicillin LB liquid medium in, spent after the violent 12~16h of shake culture of 250r/min
Endotoxin extracts plasmid kit and extracts pLVX-EF1 α-IRES-Puro-PDCoV-N plasmid, by the pLVX-EF1 α-of extraction
IRES-Puro-PDCoV-N slow virus expression plasmid and 2 helper plasmids pMD2.G and psPAX2 are used3000
Transfection reagent transfects 293T cell and carries out viral packaging (15 μ g of plasmid gross mass, the ratio of plasmid are 7:2:1), after 48h~72h
Supernatant is collected, interval 48h~72h regathers supernatant, collects 3 times altogether, the slow virus supernatant 1000r/min centrifugation of collection
10min discards bottom cell precipitation, is stored in -70 DEG C.
The determination of puromycin screening concentration: Vero cell is inoculated in 24 orifice plates, totally 11 hole, overnight incubation, cell are long
To floor space 80%~90%, the complete medium (10%FBS-DMEM) that joined puromycin successively is changed to every hole, if
The concentration gradient for setting 0,1,2,3,4,5,6,7,8,9,10 μ g/ml, every 2 days observation cell survival conditions (cell hike up as
It is dead), every the culture medium of 2 days replacement 0.75mL puromycins containing respective concentration.Screening makes cell all dead complete in 4~7 days
Minimum puromycin concentration, as screening concentration.4th day, the Vero cell in the hole containing 4 μ g/ml puromycins was extremely complete
(see Fig. 3), determine the puromycin complete medium containing 4 μ g/ml be after screening and culturing medium.
Slow-virus infection Vero cell and puromycin screening: suitable Vero cell is inoculated in 2 in 12 porocyte plates
Slow virus is infected in a hole, 1 hole, and another 1 hole when cell grows into 80%~90% floor space of covering, is discarded as control
Wherein 1 hole culture medium is cleaned 2~3 times with sterile PBS (pH7.2).The slow virus 0.5mL and 0.5mL that aspiration step (3) is collected
DMEM is uniformly mixed and is inoculated on cell, infects discarding slow virus DMEM mixed liquor after 6~8h complete medium is added, after 48h,
Change the culture medium in 2 holes into puromycin screening and culturing medium, every 2 days replacement primary screening culture mediums, until in control wells
Cell all it is dead it is complete will connect malicious hole inner cell and all produce to another 1 12 empty holes, carries out 2 times with screening and culturing medium and screens,
Cell discards 4/5 cell after covering with continues screening passage, so expands culture after 4~5 generations of screening, obtains cell line Vero/
PDCoV-N。
The PCR of recombinant cell lines Vero/PDCoV-N is identified: extracting cell line Vero/ using RNA extracts kit
The RNA of PDCoV-N is divided into 3 parts of templates for doing respective handling as PCR.1 part of direct reverse transcription is cDNA；1 part with DN ase
Carrying out reverse transcription after I processing again is cDNA；1 part of RNA template without any processing directly as PCR.With design in step (1)
Good specific primer carries out PCR amplification, and recombinant cell lines Vero/PDCoV-N is in transcriptional level successful expression as the result is shown
PDCoV-N gene (see Fig. 4).
Western-blot analyzes the expression of target gene in recombinant cell lines Vero/PDCoV-N: extracting recombinant cell lines
Vero/PDCoV-N albumen sample, while Vero cell protein sample is prepared as control, respectively with the Dan Ke of anti-Flag label protein
Grand antibody and PDCoVN protein specific monoclonal antibody are carried out as primary antibody, using the sheep anti-mouse antibody that HRP is marked as secondary antibody
Western-blot analysis, chemiluminescence develop the color the visible albumen sample with recombinant cell lines Vero/PDCoV-N preparation in 40kDa
There is specific band in left and right, and with the albumen sample of Vero cell preparation without any band (see Fig. 5 and Fig. 6), this is as the result is shown
Cell line Vero/PDCoV-N can be with successful expression PDCoVN albumen.
The application of 3 cell line Vero/PDCoV-N of embodiment
The infection pig positive serum on PDCoV infection morbidity pig farm has been made a definite diagnosis from Tianjin to acquisition and has been acquired from certain negative pig
The negative serum of field is detected, and immunohistochemical analysis is carried out with the recombinant cell lines of building, respectively with positive infection Swine serum
With negative Swine serum as primary antibody, using the HRP goat-anti pig antibody marked and FITC label goat-anti pig antibody as secondary antibody, respectively into
Row immunohistochemical staining and indirect IF staining, as the result is shown: immunohistochemical staining test in the positive
The cell line endochylema of seroreaction can dye apparent brown color, and the cell of negative serum reaction does not occur color reaction (figure
7)；It is glimmering that apparent bright green can be observed in the cell line endochylema reacted in indirect IF staining test with positive serum
Light, and the cell of negative serum reaction does not occur fluorescence (Fig. 8), which illustrates that cell line of the present invention can be with positive infection blood
It is clear that specific reaction occurs.Result above proves that the recombination Vero cell line for stablizing expression PDCoVN albumen of the invention has
Good specificity can be used for the monitoring of clinical swinery infection PDCoV.
SEQUENCE LISTING
<110>Yangzhou University
<120>a kind of the recombination Vero cell line and application of stable expression pig Delta coronavirus N protein
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213>artificial sequence
<400> 1
gaactcgaga tcatggctgc accagtagtc 30
<210> 2
<211> 50
<212> DNA
<213>artificial sequence
<400> 2
gttggatccc tacttgtcgt catcgtcttt gtagtccgct gctgattctt 50
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<400> 3
cttgtcgtca tcgtctttgt agtc 24
Claims (3)
1. a kind of recombination Vero cell line of stable expression pig Delta coronavirus N protein, which is characterized in that the cell line contains
There are pig Delta coronavirus N protein gene and resistance screening gene.
2. the recombination Vero cell line of stable expression pig Delta coronavirus N protein according to claim 1, feature
It is, it is African green monkey kidney cell which, which contains pig Delta coronavirus N protein gene and puromycin resistance gene,
Vero/PDCoV-N, deposit number are: CCTCC NO.C2018255.
3. the recombination Vero cell line of stable expression pig Delta coronavirus N protein described in claim 1 is detected in preparation
Application in pig Delta coronavirus reagent.
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| CN110361542A (en) * | 2019-07-25 | 2019-10-22 | 扬州大学 | The double-antibody sandwich elisa antigen detection kit of pig Delta coronavirus N protein |
| CN111187782A (en) * | 2020-01-20 | 2020-05-22 | 上海交通大学 | Porcine Delta coronavirus virus-like particle and preparation method and application thereof |
| CN112111465A (en) * | 2020-09-22 | 2020-12-22 | 河南牧业经济学院 | Recombinant adenovirus for expressing porcine delta coronavirus, construction method and application |
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