CN112625138A - Multi-site recombinant antigen for detecting novel coronavirus and preparation method thereof - Google Patents
Multi-site recombinant antigen for detecting novel coronavirus and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a multi-site recombinant antigen for detecting new coronavirus and a preparation method thereof, wherein S, N, E and M4 proteins are selected by a protein sequence comparison method, have extremely strong epitopes and are important protein fragments for activating an infected body to generate an antibody, the infected body can preferentially release corresponding antibodies, meanwhile, GSSSG is used for linking peptide chains to form antigen protein with multiple sites and strong immunogenicity and specificity, the recombinant protein is distinguished from other single protein sequences, fuses fragments of 4 basic proteins of the novel coronavirus, is connected in series, the recombinant antigen can specifically recognize the antibody of the serum infected by the new coronavirus, and has 4 different protein fragments, so that the sensitivity and specificity of the recombinant antigen are greatly improved, and the recombinant antigen is the best candidate antigen for detecting the new coronavirus antibody.
Description
Technical Field
The invention belongs to the field of antigen preparation, and particularly relates to a multi-site recombinant antigen for detecting a novel coronavirus and a preparation method thereof.
Background
The novel coronavirus belongs to a coronavirus of beta genus, a single-stranded positive-sense RNA virus, has an envelope, particles are round or oval, are usually polymorphic, have the diameter of 60-140nm, have certain differences with SARSr-CoV and MERSR-CoV in gene characteristics, show certain uniqueness, and show that the homology with bat SARS-like coronavirus is more than 85 percent. Therefore, the medicine has ultrahigh pathogenic rate and transmissibility, has attracted high attention of world health organization, and becomes a great threat of global public health.
The unique appearance of coronavirus is mainly reflected in the outside of viral envelope, has a similar rod-shaped particle structure, is similar to the model of the crown of king in the middle and middle European century, and is named coronavirus. The protein mainly comprises spike glycoprotein (S protein), nucleocapsid protein (N protein), envelope glycoprotein (E protein), membrane protein (M protein) and ribonucleic acid. The S protein is a rod-spherical glycosylated protein of the virus surface envelope, forms a unique coronary shape, is also a main vector for the coronavirus to invade host cells, and assists the new coronavirus to invade the cells and cause damage by combining with the AEC2 protein on the human cell surface. The NP protein is the only protein in the virus nucleus, is the protein with the most virus expression quantity, can be combined with virus RNA, and can be packaged into virus particles by envelope signals for identifying genome, and is also one of the most important antigens of coronavirus.
However, no candidate antigens suitable for the detection of new coronavirus antibodies are currently available.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a multi-site recombinant antigen for detecting a novel coronavirus, which is characterized in that the amino acid sequence of the multi-site recombinant antigen for detecting the novel coronavirus is shown as SEQ ID NO. 1.
The invention also provides a multi-site recombinant antigen for detecting the novel coronavirus, and the preparation method comprises the following steps:
(1): selecting strong antigenic determinants of S, N, E and M proteins by a protein sequence comparison method, and synthesizing a multi-site gene fragment for detecting the new coronavirus by adopting a gene cloning technology;
(2): transferring the synthesized multi-site gene fragment for detecting the new coronavirus into a pGEX-4T-2 plasmid vector and then introducing the vector into a BL21(D3) strain;
(3): and promoting bacteria to express a large amount of target protein by using IPTG (isopropyl-beta-thiogalactoside) as an inducer, and purifying by using GSH (glutathione) resin to obtain the multi-site recombinant antigen for detecting the novel coronavirus.
In any of the above embodiments, preferably, the amino acid sequence of the multi-site gene fragment for detecting the novel coronavirus is shown as SEQ ID NO. 2.
In any of the above embodiments, preferably, the pGEX-4T-2 plasmid vector has an amino acid sequence shown in SEQ ID NO. 3.
In any of the above embodiments, preferably, the four protein extraction positions are: protein 1-260: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFH AIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCE FQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFK NIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSG, respectively; e protein 1-47 MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIV; n protein 291-359: LIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLN KHIDA, respectively; m protein 20-157: WNLVIGFLFLTWICLLQFAYANRNRFLYIIK LIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPE TNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLG are provided.
The invention selects the extremely strong epitope of S, N, E, M4 proteins by the method of protein sequence comparison, which is the important protein fragment for generating antibody by activating the infected body, the infected body can release corresponding antibody preferentially, and GSSSG link peptide chain is utilized to form the antigen protein with strong immunogenicity and specificity which has multiple sites, the recombinant protein is different from other single protein sequences, the fragments of 4 basic proteins of new coronavirus are fused and connected in series, the recombinant antigen can specifically identify the antibody of serum infected by new coronavirus, and the recombinant antigen has 4 different protein fragments, thereby greatly improving the sensitivity and specificity and being the best candidate antigen for detecting the antibody of new coronavirus.
Detailed Description
In order that the invention may be further understood, the invention will now be described in detail with reference to specific examples.
The invention provides a multi-site recombinant antigen for detecting a novel coronavirus, which is characterized in that the amino acid sequence of the multi-site recombinant antigen for detecting the novel coronavirus is shown as SEQ ID NO. 1.
The invention also provides a multi-site recombinant antigen for detecting the novel coronavirus, and the preparation method comprises the following steps:
(1): selecting strong antigenic determinants of S, N, E and M proteins by a protein sequence comparison method, and synthesizing a multi-site gene fragment for detecting the new coronavirus by adopting a gene cloning technology;
(2): transferring the synthesized multi-site gene fragment for detecting the new coronavirus into a pGEX-4T-2 plasmid vector and then introducing the vector into a BL21(D3) strain;
(3): the preparation method of the protein comprises the following steps:
coli culture, 1 (glycerol bacteria): adding LB medium into 100 volumes for inoculation, and culturing overnight in a shaker (200pm) at 37 ℃; 1: continuously amplifying by adding LB culture medium with the volume of 10, and culturing for 1h by a shaking table (200pm) at the temperature of 37 ℃; adding 1mM IPTG to induce for 2h, at 30 deg.C, recovering bacteria and cracking, centrifuging to remove supernatant, and collecting precipitate; adding Wash Buffer, uniformly mixing by using a micropipette, centrifuging for 10000 revolutions for 10 minutes, and cleaning twice; adding (20 bacteria liquid: 1) balance liquid into the centrifugal precipitate, reacting in a shaking table at 4 ℃ overnight (or at room temperature for 1h), and fully dissolving the precipitate (clearing); centrifuging for 10000 revolutions for 10 minutes, and taking a supernatant; equilibrate the resin column containing GST affinity with equilibrating solution, wash repeatedly 2 times; adding a buffer solution containing a sample to the well-balanced resin column; after the sample loading is finished, continuously leaching the sample to a baseline by using an equilibrium buffer solution; the column was equilibrated with 10 bed volumes of PreScission Protease digestion buffer. The components of the enzyme digestion buffer solution of PreScission Protease are 50mM Tris-HCl, 150mM NaCl, 1mM ED TA, 1mM DTT and pH7.5; preparing PreScission Protease: about 20U PreScission Protease was used per mg GST tag protein. 160U PreScission Protease is needed for 8mg GST tag protein, and the GST tag protein is diluted to the volume which is 1ml and is the same as that of the gel column by PreScission enzyme digestion buffer solution; introducing diluted 1ml PreScission Protease into a purification column, and keeping the temperature at 4 ℃ for 4-8h (in order to ensure complete enzyme digestion, enzyme digestion can be carried out overnight at 4 ℃); washing with PreScission Protease enzyme digestion buffer solution with the volume of 1 time of the bed, repeating the washing for three times, and collecting the washing liquid for each time. The eluate contains the target new coronavirus recombinant protein with the GST tag removed, and the GST tag and the PreScission Protease with the GST tag are still bound on the gel column.
In any of the above embodiments, preferably, the amino acid sequence of the multi-site gene fragment for detecting the novel coronavirus is shown as SEQ ID NO. 2.
In any of the above embodiments, preferably, the pGEX-4T-2 plasmid vector has an amino acid sequence shown in SEQ ID NO. 3.
In any of the above embodiments, preferably, the four protein extraction positions are: protein 1-260: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFH AIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCE FQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFK NIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSG, respectively; e protein 1-47 MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIV; n protein 291-359: LIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLN KHIDA, respectively; m protein 20-157: WNLVIGFLFLTWICLLQFAYANRNRFLYIIK LIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPE TNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLG are provided.
The invention selects the extremely strong epitope of S, N, E, M4 proteins by the method of protein sequence comparison, which is the important protein fragment for generating antibody by activating the infected body, the infected body can release corresponding antibody preferentially, and GSSSG link peptide chain is utilized to form the antigen protein with strong immunogenicity and specificity which has multiple sites, the recombinant protein is different from other single protein sequences, the fragments of 4 basic proteins of new coronavirus are fused and connected in series, the recombinant antigen can specifically identify the antibody of serum infected by new coronavirus, and the recombinant antigen has 4 different protein fragments, thereby greatly improving the sensitivity and specificity and being the best candidate antigen for detecting the antibody of new coronavirus.
The use scheme is as follows:
case (2):
preparing an emulsion (200-400 mu l/mouse) of PBS (phosphate buffer solution) with the same volume containing 25-100 mu G of antigen and Freund's complete adjuvant, and injecting the emulsion into the abdominal cavity of a mouse by using a 22G needle; after 1 week, re-injecting the emulsion (200-400 mu l) prepared by PBS containing 10-50 mu g of antigen and incomplete Freund's adjuvant in the same volume into the abdominal cavity; after 1 week, 100-; the collected blood, from which serum was extracted, was filled into another microcentrifuge tube. Serum antibody titers were determined by ELISA.
Generally, the injected mouse can form an anti-new coronavirus antigen specific antibody, the antibody titer can reach not less than 1/1000 through detection, the antigen can be used as an in vitro detection antibody raw material, and meanwhile, the in vivo antibody of the mouse can be extracted for in vitro antigen diagnosis of the new coronavirus.
The new coronavirus multi-site recombinant protein contains most of epitopes of new coronaviruses, so that the new coronavirus multi-site recombinant protein can effectively activate organisms and release corresponding antibodies, can be used as a raw material for detecting new coronaviruses formed in blood, can be used as an antigen for detection in aspects of ELISA (enzyme-linked immunosorbent assay), colloidal gold, immunofluorescence and the like, is specially used for detecting the new coronaviruses, and can be used as a raw material for preparing related antibodies (mice, rabbits and the like).
It will be understood by those skilled in the art that the multi-site recombinant antigen for coronavirus detection and the method for preparing the same according to the present invention include the contents of the above description of the present invention, are limited to space and are not described in detail for the sake of brevity. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Suzhou Nowegian Baiao Biotechnology Ltd
<120> multi-site recombinant antigen for detecting novel coronavirus and preparation method thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6533
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg 60
gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt 120
tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc 180
tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca 240
cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc 300
aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc 360
gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc 420
ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata 480
tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc 540
ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact 600
ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag 660
atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt 720
tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa 780
aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat 840
ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc 900
atcctccaaa atcggatctg gttccgcgtg gatccaatgt tcgtttttct ggttctgctg 960
ccattagttt cctctcagtg cgttaatctg accactcgta ctcaactgcc gccggcatat 1020
accaatagtt ttacccgcgg cgtctattat ccggataaag tctttcgcag cagcgttctg 1080
catagcaccc aggacctgtt tctgccgttt ttcagcaacg tcacctggtt tcacgcgatt 1140
cacgttagcg gtaccaacgg taccaaacgt ttcgataacc cggttctgcc gtttaacgac 1200
ggggtttact tcgcgagcac cgagaaaagc aacatcattc gcggttggat ttttggtacc 1260
accctggata gcaaaaccca gagcctgctg attgtcaaca acgcgaccaa cgtcgtcatc 1320
aaagtctgcg agttccagtt ctgcaacgat ccgtttctgg gcgtctacta ccacaaaaac 1380
aacaaatcct ggatggagag cgaatttcgc gtttatagca gcgcaaacaa ctgcaccttc 1440
gaatacgtca gccaaccgtt tctgatggat ctggaaggca aacagggcaa cttcaaaaac 1500
ctgcgcgagt tcgtcttcaa aaacatcgac ggctacttca aaatctacag caaacacacc 1560
ccgatcaatc tggttcgcga tctgccgcaa ggtttttctg cactggaacc gctggtagat 1620
ctgccgattg gcatcaacat cacccgcttt cagaccctgc tggcactgca tcgtagttat 1680
ctgaccccgg gcgattcttc ctctggtatg tatagtttcg ttagcgaaga aaccggcacc 1740
ctgattgtta attctgtctt gctgtttctg gcgtttgtcg tttttctgct ggttaccctg 1800
gcgattctga ccgctctgcg tctgtgcgcg tactgttgta atatagtcgg ttccagttca 1860
ggtctgattc gtcagggtac cgactacaaa cactggccgc aaatcgcaca atttgcaccg 1920
tctgcatctg ccttctttgg catgtctcgt attggcatgg aagttacccc gtctggtacc 1980
tggctgacct ataccggcgc aattaaactg gacgacaaag acccgaactt caaagaccag 2040
gtcatcctgc tgaacaaaca tatcgacgcg ggtagtagca gcggttggaa tctggttatc 2100
ggctttctgt tcctgacctg gatctgtctg ctgcaatttg cgtacgcgaa tcgtaaccgc 2160
tttctgtaca tcatcaaact gatcttcctg tggctgctgt ggccagtaac tctggcttgt 2220
tttgttctgg cggcggttta tcgtatcaac tggattaccg gcggtattgc cattgcaatg 2280
gcatgtctgg tcggtctgat gtggctgagc tatttcattg cgagctttcg tctgtttgct 2340
cgtacccgta gtatgtggag ctttaacccg gaaaccaaca ttctgctgaa cgttccgctg 2400
cacggtacca ttctgacccg tccgctgctg gaatctgaac tggttattgg cgcagttatt 2460
ttgcgtggtc atcttcgcat tgctggtcat catctgggtt aagaattccc gggtcgactc 2520
gagcggccgc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa 2580
aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 2640
agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 2700
acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg cctcgtgata 2760
cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 2820
tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 2880
tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 2940
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 3000
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 3060
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 3120
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 3180
cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 3240
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 3300
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 3360
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 3420
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 3480
cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 3540
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 3600
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 3660
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 3720
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 3780
tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 3840
ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 3900
accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 3960
aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 4020
ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 4080
gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 4140
ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 4200
ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 4260
ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 4320
gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 4380
cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 4440
cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 4500
cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 4560
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 4620
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 4680
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 4740
gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataaat 4800
tccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag 4860
agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc 4920
ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa 4980
acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca 5040
caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg 5100
cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc 5160
gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat 5220
cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc 5280
attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag 5340
acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat 5400
ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg 5460
gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata 5520
gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg 5580
aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca 5640
atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac 5700
gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt 5760
cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg 5820
aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat 5880
acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 5940
tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 6000
ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 6060
ataacaattt cacacaggaa acagctatga ccatgattac ggattcactg gccgtcgttt 6120
tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 6180
cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 6240
tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg 6300
aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc 6360
agatgcacgg ttacgatgcg cccatctaca ccaacgtaac ctatcccatt acggtcaatc 6420
cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg 6480
aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttgga att 6533
<210> 2
<211> 1563
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgttcgttt ttctggttct gctgccatta gtttcctctc agtgcgttaa tctgaccact 60
cgtactcaac tgccgccggc atataccaat agttttaccc gcggcgtcta ttatccggat 120
aaagtctttc gcagcagcgt tctgcatagc acccaggacc tgtttctgcc gtttttcagc 180
aacgtcacct ggtttcacgc gattcacgtt agcggtacca acggtaccaa acgtttcgat 240
aacccggttc tgccgtttaa cgacggggtt tacttcgcga gcaccgagaa aagcaacatc 300
attcgcggtt ggatttttgg taccaccctg gatagcaaaa cccagagcct gctgattgtc 360
aacaacgcga ccaacgtcgt catcaaagtc tgcgagttcc agttctgcaa cgatccgttt 420
ctgggcgtct actaccacaa aaacaacaaa tcctggatgg agagcgaatt tcgcgtttat 480
agcagcgcaa acaactgcac cttcgaatac gtcagccaac cgtttctgat ggatctggaa 540
ggcaaacagg gcaacttcaa aaacctgcgc gagttcgtct tcaaaaacat cgacggctac 600
ttcaaaatct acagcaaaca caccccgatc aatctggttc gcgatctgcc gcaaggtttt 660
tctgcactgg aaccgctggt agatctgccg attggcatca acatcacccg ctttcagacc 720
ctgctggcac tgcatcgtag ttatctgacc ccgggcgatt cttcctctgg tatgtatagt 780
ttcgttagcg aagaaaccgg caccctgatt gttaattctg tcttgctgtt tctggcgttt 840
gtcgtttttc tgctggttac cctggcgatt ctgaccgctc tgcgtctgtg cgcgtactgt 900
tgtaatatag tcggttccag ttcaggtctg attcgtcagg gtaccgacta caaacactgg 960
ccgcaaatcg cacaatttgc accgtctgca tctgccttct ttggcatgtc tcgtattggc 1020
atggaagtta ccccgtctgg tacctggctg acctataccg gcgcaattaa actggacgac 1080
aaagacccga acttcaaaga ccaggtcatc ctgctgaaca aacatatcga cgcgggtagt 1140
agcagcggtt ggaatctggt tatcggcttt ctgttcctga cctggatctg tctgctgcaa 1200
tttgcgtacg cgaatcgtaa ccgctttctg tacatcatca aactgatctt cctgtggctg 1260
ctgtggccag taactctggc ttgttttgtt ctggcggcgg tttatcgtat caactggatt 1320
accggcggta ttgccattgc aatggcatgt ctggtcggtc tgatgtggct gagctatttc 1380
attgcgagct ttcgtctgtt tgctcgtacc cgtagtatgt ggagctttaa cccggaaacc 1440
aacattctgc tgaacgttcc gctgcacggt accattctga cccgtccgct gctggaatct 1500
gaactggtta ttggcgcagt tattttgcgt ggtcatcttc gcattgctgg tcatcatctg 1560
ggt 1563
<210> 3
<211> 4970
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg 60
gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt 120
tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc 180
tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca 240
cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc 300
aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc 360
gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc 420
ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata 480
tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc 540
ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact 600
ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag 660
atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt 720
tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa 780
aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat 840
ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc 900
atcctccaaa atcggatctg gttccgcgtg gatccccagg aattcccggg tcgactcgag 960
cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac 1020
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 1080
agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc 1140
cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc 1200
ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 1260
cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 1320
ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 1380
agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 1440
tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 1500
gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 1560
gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 1620
gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 1680
gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 1740
agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 1800
ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 1860
cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 1920
gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 1980
cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 2040
gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 2100
ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 2160
acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 2220
ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 2280
aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 2340
aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 2400
ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 2460
ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 2520
actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 2580
caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 2640
gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 2700
ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 2760
cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 2820
cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 2880
acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 2940
ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 3000
gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 3060
tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 3120
accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 3180
cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc 3240
gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt 3300
caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt 3360
gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg 3420
cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa 3480
caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac 3540
gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg 3600
gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt 3660
ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt 3720
gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca 3780
cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg 3840
gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg 3900
cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg 3960
gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat 4020
gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg 4080
cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac 4140
gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc 4200
ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag 4260
ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg 4320
caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 4380
cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 4440
accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 4500
acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac 4560
aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 4620
ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 4680
gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa 4740
gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga 4800
tgcacggtta cgatgcgccc atctacacca acgtaaccta tcccattacg gtcaatccgc 4860
cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa 4920
gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt 4970
Claims (5)
1. The multi-site recombinant antigen for detecting the new coronavirus is characterized in that the amino acid sequence of the multi-site recombinant antigen for detecting the new coronavirus is shown as SEQ ID NO. 1.
2. The multi-site recombinant antigen for detecting a novel coronavirus according to claim 1, which is prepared by the following steps:
(1): selecting strong antigenic determinants of S, N, E and M proteins by a protein sequence comparison method, and synthesizing a multi-site gene fragment for detecting the new coronavirus by adopting a gene cloning technology;
(2) transferring the synthesized multi-site gene fragment for detecting the new coronavirus into a pGEX-4T-2 plasmid vector and then introducing the vector into a BL21(D3) strain;
(3) and promoting bacteria to express a large amount of target protein by using IPTG (isopropyl-beta-thiogalactoside) as an inducer, and purifying by using GSH (glutathione) resin to obtain the multi-site recombinant antigen for detecting the novel coronavirus.
3. The multi-site recombinant antigen for detecting new coronavirus according to claim 2, wherein the amino acid sequence of the multi-site gene fragment for detecting new coronavirus is shown as SEQ ID No. 2.
4. The multi-site recombinant antigen for detecting a novel coronavirus according to claim 2, wherein the amino acid sequence of the pGEX-4T-2 plasmid vector is represented by SEQ ID No. 3.
5. The multi-site recombinant antigen for detecting new coronavirus according to claim 2, wherein the four protein extraction sites are: protein 1-260: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSG, respectively; e protein 1-47 MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIV; n protein 291-359: LIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDA, respectively; m protein 20-157: WNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLG are provided.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116574195A (en) * | 2023-07-07 | 2023-08-11 | 北京市疾病预防控制中心 | Novel coronavirus antibody and application thereof in detection of oral liquid without transmission risk |
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| CN1990042A (en) * | 2005-05-31 | 2007-07-04 | 中国人民解放军第三军医大学第一附属医院 | SARS coronary virus resistant cell vaccine and its uses |
| CN111239394A (en) * | 2020-03-09 | 2020-06-05 | 四川省人民医院 | Novel coronavirus antibody rapid detection kit based on mixed antigens |
| CN111303254A (en) * | 2020-02-20 | 2020-06-19 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) antigen detection kit |
| CN111551713A (en) * | 2020-05-15 | 2020-08-18 | 中国科学院过程工程研究所 | COVID-19 virus antibody detection microsphere, preparation method thereof and kit containing microsphere |
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| CN1990042A (en) * | 2005-05-31 | 2007-07-04 | 中国人民解放军第三军医大学第一附属医院 | SARS coronary virus resistant cell vaccine and its uses |
| CN111303254A (en) * | 2020-02-20 | 2020-06-19 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) antigen detection kit |
| CN111239394A (en) * | 2020-03-09 | 2020-06-05 | 四川省人民医院 | Novel coronavirus antibody rapid detection kit based on mixed antigens |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116574195A (en) * | 2023-07-07 | 2023-08-11 | 北京市疾病预防控制中心 | Novel coronavirus antibody and application thereof in detection of oral liquid without transmission risk |
| CN116574195B (en) * | 2023-07-07 | 2023-09-29 | 北京市疾病预防控制中心 | Novel coronavirus antibody and application thereof in detection of oral liquid without transmission risk |
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