CN113846154B - Application of rs12252 polymorphism in detection of novel coronavirus antibody - Google Patents

Application of rs12252 polymorphism in detection of novel coronavirus antibody Download PDF

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CN113846154B
CN113846154B CN202111187219.3A CN202111187219A CN113846154B CN 113846154 B CN113846154 B CN 113846154B CN 202111187219 A CN202111187219 A CN 202111187219A CN 113846154 B CN113846154 B CN 113846154B
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覃岭
赵艳
张永宏
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Beijing Youan Hospital
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Abstract

The invention discloses application of rs12252 polymorphism in detection of a novel coronavirus antibody. The invention discovers that the number of 4 times of the neutralizing antibodies of CC genotype crowd at the rs12252 locus is obviously higher than CT genotype and TT genotype after 90 days of inoculation of the human with the novel coronavirus vaccine. The specific antibody level of the novel coronavirus vaccine against the novel coronavirus in the CC genotype group is higher. The invention can be used for detecting the titer level of a specific antibody generated against a new coronavirus after the new coronavirus vaccine is inoculated to a human, screening the human with a low antibody level after the new coronavirus vaccine is inoculated, and detecting the resistance to the new coronavirus after the new coronavirus vaccine is inoculated to the human.

Description

Application of rs12252 polymorphism in detection of novel coronavirus antibody
Technical Field
The invention relates to application of polymorphism rs12252 in detecting a novel coronavirus antibody in the biomedical field.
Background
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to variation of a single nucleotide in the genome, which is the smallest unit of variation, which is a form of variation formed by substitution, transversion, insertion or deletion of a single nucleotide pair. Single nucleotide polymorphisms are a high-density genetic markers on the genome, and the number of SNPs found in the human genome exceeds 3000 tens of thousands. As the third generation genetic markers SNP are numerous in number, densely distributed and easy to detect, the method is an ideal genotyping target.
rs12252 is a single SNP site for a two-position polymorphism on human chromosome 11p5.5, the variation being a transition (T/C, A/G on its complementary strand), located in the interferon inducible transmembrane protein 3 (IFITM 3) gene.
Disclosure of Invention
The invention aims to solve the technical problem of how to detect the titer level of a specific antibody generated against a new coronavirus after the new coronavirus vaccine is inoculated to a human, or to screen the human with a low antibody level after the new coronavirus vaccine is inoculated to the human, or to detect the resistance to the new coronavirus after the new coronavirus vaccine is inoculated to the human.
In order to solve the technical problems, the invention firstly provides any one of the following applications 1-3:
1. use of a substance that detects a polymorphism or genotype of rs12252 in the human genome for the preparation of a product for evaluating or aiding in the evaluation of the titer level of specific antibodies raised against a novel coronavirus after vaccination of a human with the novel coronavirus vaccine;
2. use of a substance that detects the polymorphism or genotype of rs12252 in the human genome for the preparation of a product for screening or assisted screening of a human with a low antibody level after vaccination with a new coronavirus vaccine; the antibody is a specific antibody aiming at the novel coronavirus;
3. use of a substance for detecting the polymorphism or genotype of rs12252 in the human genome for the preparation of a product for detecting or aiding in the detection of resistance to a new coronavirus after vaccination of a human with the new coronavirus vaccine.
rs12252 is the SNP site for one of the two allelic polymorphisms on human chromosome 11p5.5, the variation being a shift (T/C, A/G on its complementary strand).
In the above application, the person may be a person aged 25-59 years. The human may be a human vaccinated with the novel coronavirus vaccine. The human may in particular be a human vaccinated with the new coronavirus for 14-90 days.
In the above application, the novel coronavirus vaccine may be a vaccine for preventing pneumonia caused by a novel coronavirus.
In the application, the novel coronavirus vaccine is a novel coronavirus inactivated vaccine (Vero cell). The novel coronavirus vaccine can be specifically a novel coronavirus inactivated vaccine (Vero cells) produced by vitamin technology limited company in Beijing.
In the above application, the rs12252 genotype is CC, CT or TT. Wherein CC is homozygous for rs12252 site C, TT is homozygous for rs12252 site T, and CT is heterozygous for rs12252 site T and C.
In the application, the total immunization of the human with the novel coronavirus vaccine is carried out for 90 days, and the number of 4 times of the neutralizing antibodies of the CC genotype crowd is obviously higher than that of CT genotype and TT genotype. The specific antibody level of the novel coronavirus vaccine against the novel coronavirus in the CC genotype group is higher.
In the above application, the detection of the polymorphism or genotype of rs12252 in the human genome may be specifically determined by detecting the nucleotide species of rs12252.
In the above applications, the substance that detects the polymorphism or genotype of rs12252 in the human genome may be reagents and/or instrumentation required to determine the polymorphism or genotype of rs12252 by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography, SNP chip, taqMan probe technology and Sequenom MassArray technology. Among the reagents and/or instrumentation required to determine the polymorphism or genotype of rs12252 using Sequenom MassArray techniques include PCR primer pairs, extension primers based on single base extension reactions, phosphatases (e.g., shrimp alkaline phosphatase (shrimp alkaline phosphatase, SAP)), resins, chips, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight, matrix-assisted laser desorption ionization time-of-flight mass spectrometry), and other reagents and instrumentation required by Sequenom MassArray techniques; reagents and/or instrumentation required to determine the polymorphism or genotype of rs12252 using restriction enzyme fragment length polymorphism include PCR primer pairs and restriction enzymes; SNP chips include chips based on nucleic acid hybridization reactions, chips based on single base extension reactions, chips based on allele-specific primer extension reactions, chips based on "one-step" reactions, chips based on primer ligation reactions, chips based on restriction enzyme reactions, chips based on protein DNA binding reactions, and chips based on fluorescent molecule DNA binding reactions.
In the above application, the substance for detecting the polymorphism or genotype of rs12252 in human genome may comprise a PCR primer pair for amplifying a genomic DNA fragment including rs12252 and/or a restriction endonuclease MscI. The substance that detects the polymorphism or genotype of rs12252 in the human genome may also be only a PCR primer pair that amplifies genomic DNA fragments including rs12252 and/or restriction enzyme MscI.
In one embodiment of the invention, the polymorphism and genotype of rs12252 is determined using restriction enzyme fragment length polymorphism. The PCR primer pair has no special requirement on the sequence, so long as the PCR primer pair can amplify genome DNA fragments including rs12252, and the PCR primer pair can be specifically single-stranded DNA shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table. The restriction enzyme may specifically be MscI.
In the above application, the product for evaluating the titer level of the specific antibody against the novel coronavirus after the human is vaccinated with the novel coronavirus, the product for screening the human with a low antibody level after the human is vaccinated with the novel coronavirus, and the product for detecting the resistance of the human to the novel coronavirus after the human is vaccinated with the novel coronavirus may be a reagent, a kit or a system. In such applications, the system may include reagents, kits, and/or instruments.
In the above application, the product for evaluating the titer level of the specific antibody against the new coronavirus after the human is vaccinated with the new coronavirus, the product for screening the human with the low antibody level after the human is vaccinated with the new coronavirus, and the product for detecting the resistance to the new coronavirus after the human is vaccinated with the new coronavirus may comprise the substance for detecting the polymorphism or genotype of rs12252 in the human genome, and may further comprise a system for quantitatively detecting the antibody level against the new coronavirus. The system for quantitatively detecting the antibody level against the novel coronavirus may be reagents and/or instruments required for detecting the antibody level against the novel coronavirus by an enzyme-linked immune reaction.
Herein, the antibody level against the new coronavirus may be an antibody level against the new coronavirus in plasma.
The invention can be used for detecting the titer level of a specific antibody generated against a new coronavirus after the new coronavirus vaccine is inoculated to a human, screening the human with a low antibody level after the new coronavirus vaccine is inoculated, and detecting the resistance to the new coronavirus after the new coronavirus vaccine is inoculated to the human.
Drawings
FIG. 1 changes in neutralizing antibody titers at different time points of vaccination with the novel coronaries. Panel B shows that each genotype was 21 days after the first dose, 14 days after the second dose, and 90 days after the second dose, before vaccination, in order from left to right.
Figure 2 compares fold increase in neutralizing antibodies after vaccination with the new corona vaccine.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
Example 1,
rs12252 is a single SNP site for a two-position polymorphism on human chromosome 11p5.5, the variation being a shift (T/C, A/G on its complementary strand), the site being located in the IFITM3 gene, designated IFITM3rs 12252. The rs12252 genotype is CC, CT or TT. Wherein CC is homozygous for rs12252 site C, TT is homozygous for rs12252 site T, and CT is heterozygous for rs12252 site T and C.
1. Subject and sample
129 healthy volunteers (uninfected with the novel coronavirus) vaccinated with the novel coronavirus inactivated vaccine (Vero cells) from vitamin technologies limited in beijing, were aged between 25-59 years.
Genomic DNA of each person was extracted.
2. Genotyping of rs12252 locus
(1) Amplification of nucleotide fragments containing SNP loci
Primers were designed based on the flanking sequences of rs12252, forward primer F:5'-GAAAAGGAAACTGTTGAGAACCGAA-3' (SEQ ID NO. 1), reverse primer R:5'-GAGCCTCCTCCTAAACCTGCAC-3' (SEQ ID NO. 2) and amplifying the nucleotide fragment of the SNP to be detected, wherein the PCR product is SEQ ID NO.3 in the sequence table. The rs12252 locus is located at 140bp of SEQ ID NO.3, where the nucleotide is C or T, and when the nucleotide is T, it can be recognized by MscI endonuclease (recognition sequence is TGG CCA).
Wherein, the PCR reaction system is 50 μl: 150-200 ng/. Mu.l of human genomic DNA 1. Mu.l, 10. Mu.M primers F and R2. Mu.l each, premix Taq HS 25. Mu.l, the balance ddH 2 O; wherein, the Premix Taq HS is a product of TaKaRa company (product number: DR 028A). Premix Taq Hot Start Version (TaKaRa Co.).
The PCR reaction conditions were: 95 ℃ for 10 minutes; 95℃for 60 seconds, 55℃for 30 seconds, 72℃for 30 seconds, 35 cycles; and at 72℃for 5 minutes.
(2) PCR-RFLP with appropriate endonuclease selection for nucleotide 142
The PCR product was digested with MscI, and the reaction system was 20. Mu.l: PCR amplification product 10. Mu.l, quick digestion MscI enzyme (Fast Digest MScI, fermentas Co.) 1. Mu.l, 10 XFastDiget Green buffer 2. Mu.l, ddH without nuclease 2 O17. Mu.l. The reaction conditions are as follows: and (3) carrying out water bath at 37 ℃ and enzyme digestion for 30min.
Then, the resulting digested product was subjected to electrophoresis in 2% agarose gel, stained with ethidium bromide, and observed in a gel imaging system.
And (3) glue preparation: washing the vessel contacted with the gel with distilled water, naturally airing at room temperature, pouring 2% agarose gel containing Golden View into a template, inserting a comb with 13 holes, and pulling out the comb after the gel is naturally polymerized, thus obtaining 2% agarose gel containing 13 holes.
Electrophoresis: to 15. Mu.l of the digested product was added 3. Mu.l of a 6 Xloading buffer, and the sample was subjected to loading detection, 100V constant pressure, and electrophoresis for 2 hours.
The genotype of the locus in the test population was determined based on the results of PCR-RFLP. When the 140bp nucleotides of the PCR product are all C, the MscI endonuclease cannot recognize the locus, namely the PCR product cannot be cut, the gel has only one band, the length is 562bp, and the genotype is marked as CC type; when the nucleotides at 140bp are T, the PCR amplified fragment can be cut into two fragments, one fragment is 420bp, the other fragment is 142bp, and the genotype is marked as TT type; when the 140bp position contains both C and T, namely three bands are contained in gel electrophoresis, the lengths are about 562bp, 420bp and 142bp respectively, and the genotype is marked as heterozygous CT type. (FIG. 1)
The results showed that of the 129 volunteers, 38 human CC genotype, 59 human CT genotype, 32 human TT genotype, and the different IFITM3rs12252 gene polymorphism populations had no significant differences between age and gender, table 1.
TABLE 1 comparison of age and sex in different IFITM3rs12252 Gene polymorphism populations
3. Detection of neutralizing antibodies
The detection of neutralizing antibodies was performed by taking peripheral blood plasma before each volunteer was vaccinated, on day 21 of vaccination with the first injection vaccine, on day 14 of full-course vaccine immunization (i.e., on day 14 of vaccination with the second injection vaccine), on day 90 of full-course vaccine immunization (i.e., on day 90 of vaccination with the second injection vaccine), and using a neutralizing antibody detection kit (Beijing heat-clearing Biotechnology Co., ltd., lot number: 21010115). Experimental principle: based on the magnetic particle chemiluminescence immunoassay technology, a competition method is adopted to detect novel coronavirus neutralizing antibodies in a plasma sample.
The results show that: the neutralizing antibody maintaining time of the novel crown vaccine inoculated to the CC genotype group of 25-59 years old is longer than that of the CT/TT genotype group. The median of neutralizing antibody titers of up to 32 (16,128) at 14 days after full-range vaccine immunization of 129 volunteers was significantly higher than 4 (4, 4) at 21 days after first injection vaccine inoculation and 8 (8, 16) at 90 days after full-range immunization, i.e. neutralizing antibody levels were significantly reduced after 90 days after new crown vaccine inoculation (fig. 1, a); the dynamic change condition of the neutralizing antibody titer at different time points of different IFITM3rs12252 gene polymorphism groups vaccinated is consistent with the change trend of the whole group. The median of the neutralizing antibody titer of 14 days after the whole vaccine immunization of the CC genotype group reaches the highest 32 (16,128), which is obviously higher than 4 (4, 4) of 21 days after the first injection vaccine inoculation and 16 (8, 32) of 90 days after the whole vaccine immunization; the median of neutralizing antibody titer of 14 days after full-course vaccine immunization of CT genotype population reaches the highest 32 (16, 64), which is obviously higher than 4 (4, 4) of 21 days after first injection vaccine inoculation and 8 (8, 16) of 90 days after full-course immunization; the median of neutralizing antibody titer of 14 days after full-course vaccine immunization of TT genotype group reached the highest 32 (20, 128), which is significantly higher than 4 (4, 4) of 21 days after first injection vaccine inoculation and 8 (8, 16) of 90 days after full-course immunization, as shown in FIGS. 1 and B; the median of neutralizing antibody titers in the CC genotype population was 16 (8, 32) 90 days after full immunization, higher than that of CT genotype 8 (4, 8) and TT genotype 8 (4, 8), but no significant differences were seen, with p-values of 0.1239 (fig. 1, c), respectively.
The results also show that: the median of the increase of the neutralizing antibody titer of the populations of CC, CT and TT genotypes were 1 (1, 1) 21 days after the first injection vaccine, and there was no significant difference between them (FIG. 2, A); the median fold increase of neutralizing antibodies in populations with CC, CT and TT genotypes was 8 (4,32), 8 (4, 16) and 8 (5,32), respectively, 14 days after full immunization, with no significant differences (fig. 2, b); 90 days after full immunization, the fold increase in neutralizing antibodies in the CC genotype population was 4 (2, 8) higher than CT (2, p= 0.0553) and TT (2, p=0.1125), but no significant differences were seen (fig. 2, c); the percentage of neutralizing antibodies 4 multiplied by the long people with the CC genotype is 0% after the first injection vaccine is inoculated, the percentage is obviously lower than that of CT genotype (7%, p=0.0104), and no obvious difference exists between CC and TT and between CT and TT (figures 2 and D); 14 days after full immunization, the percentage of neutralizing antibodies 4 multiplied by the population with CC genotype was 89%, which is higher than CT genotype (81%) and TT genotype (78%), with no significant difference (FIG. 2, E); 90 days after full immunization, the percentage of neutralizing antibodies 4 multiplied by the population of CC genotypes was 63% (24/38), significantly higher than the CT genotypes (41%, p=0.0003, 24/59) and TT genotypes (41%, p=0.0003, 13/32), see figure 2,F.
Fold increase in neutralizing antibody titer: neutralizing antibody titer at various time points post immunization/neutralizing antibody titer before immunization.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
<110> affiliated Beijing you an Hospital of university of capital medical science
Application of polymorphism of <120> rs12252 in detection of novel coronavirus antibody
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cccagtaacc cgaccgccgc tggtcttcgc tggacaccat gaatcacact gtccaaacct 120
tcttctctcc tgtcaacagy ggccagcccc ccaactatga gatgctcaag gaggagcacg 180
aggtggctgt gctgggggcg ccccacaacc ctgctccccc gacgtccacc gtgatccaca 240
tccgcagcga gacctccgtg cccgaccatg tcgtctggtc cctgttcaac accctcttca 300
tgaacccctg ctgcctgggc ttcatagcat tcgcctactc cgtgaaggtg cgtatggccc 360
cagggaatgc tcagagggtg ccgctgagcc tggagctcca cctgcccaca tgctgcctgg 420
ggtggggact tgtgtgtccc tgtgactgtg agtttgtgtg cacctctgtc ccgtgtgtgc 480
ccacgtcagt ggctttgtct gtgtgatctg tgtgtgtgtg tggcttgggg aatctgccca 540
gtgcaggttt aggaggaggc tc 562

Claims (1)

1. Use of a substance that specifically detects the genotype of rs12252 in the genome of a human, said human being aged 25-59 years old, said genotype of rs12252 being CC, CT or TT genotype, for the preparation of a product for evaluating or aiding in the evaluation of the titer level of specific antibodies raised against a novel coronavirus after vaccination of a human with a novel coronavirus inactivated vaccine, wherein the CC genotype neutralizing antibody has a longer duration than the CT or TT genotype.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108330196A (en) * 2018-02-06 2018-07-27 首都医科大学附属北京佑安医院 Application of the polymorphism of rs12252 in detecting Antibody of Influenza

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108330196A (en) * 2018-02-06 2018-07-27 首都医科大学附属北京佑安医院 Application of the polymorphism of rs12252 in detecting Antibody of Influenza

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Abhijit Pati等.Minor allel of interferon-induced transmembrane protein 3 polymorphism (rs12252) is covered against severe acute respiratory syndrome coronavirus 2 infection and mortality: a worldwide epidemiological investigation.the journal of infectious diseases.2021,175-178. *
Fahad S. Mohammed等.The interferon-induced transmembrane protein 3-rs12252 allele may predict COVID-19 severity among ethnic minorities.《frontiers in Genetics》.2021,第12卷第4页右栏第2段. *
Ling Qin等.high level antibody response to pandemic influenza H1N1/09 virus is associated with interferon-induced transmembrane protein-3 rs12252-CC in young adults.frontiers in cellular and infection microbiology.2018,第8卷1-6. *

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