CN111662990A - Method and primer pair for quantitatively detecting different levels of swine fever antibody - Google Patents

Abstract

The invention discloses a method and a primer pair for quantitatively detecting different levels of swine fever antibodies, wherein the primer is designed by utilizing Oligo6.0 software and MethyPrimer online software, and Bisulfit sequencing is used to be combined with a classical Bisulfite sequencing method for verification, so that the levels of different swine fever antibodies can be rapidly, accurately and efficiently determined; the method provided by the invention comprises the steps of carrying out PCR amplification on genome DNA treated by bisulfite of the pig to be detected by using the primer pair, and then judging whether the pig to be detected is a CD4gene promoter region which is hypermethylated or moderately methylated according to a pyrosequencing result, wherein the swine fever antibody level of the hypermethylated pig is higher than that of the moderately methylated pig; the swine fever antibody level is detected by using test paper, and the method provided by the invention is convenient and accurate to use, has high sensitivity, and can be used for detecting the swine fever antibody level of Changbai and Dabai swine fever antibodies.

Description

Method and primer pair for quantitatively detecting different levels of swine fever antibody

Technical Field

The invention relates to the field of epigenetics and molecular immunology, in particular to a method and a primer pair for quantitatively detecting different levels of swine fever antibodies.

Background

China is one of the most pig-raising countries in the world, and the pig raising industry plays a significant role in the animal husbandry in China. However, in actual production, classical Swine Fever Virus (Classic Swine river Virus) infects Swine herds by infecting a plurality of viruses such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), influenza Virus (SIV) and pseudorabies Virus (PRV) individually or in combination. These viruses often destroy the immune function of host cells by using immune-competent lymphocytes, macrophages, endothelial cells, and some tissue epithelial cells as important target cells. The virus such as hog cholera generally causes immunosuppression of the body, which is expressed by inducing apoptosis of tissue inherent cells and lymphocytes, inhibiting production of type I interferon, inducing leucopenia, and finally causing immunosuppression of the body of the pig with the virus. In this case, the pigs are on the one hand in sub-health status, slow in growth and even onset of disease, eventually leading to a breakdown of the immune system; on the other hand, on the basis of tissue degeneration caused by infection, the exudate comprises natural killer cells (NK cells), monocytes, specific T cells and the like, which form a network system with signals and effects to comprehensively inhibit damage caused by virus infection. The fundamental reason is that the gene expression of host cells is changed after virus infection, and the change is beneficial to the reproduction of pathogens and is beneficial to the elimination of pathogens initiated by hosts.

How to quantitatively detect whether the hog cholera is successfully immunized becomes important in the research field. Most of the immune studies on swine fever are related immune genes such as cytokines and cell surface receptors, interferon response factors essential for innate resistance to infection, T cell differentiation related genes and the like. The pathogenesis of the swine fever is researched by using an expression profiling method to be related to the cell response of a host, and the expression quantity of some genes is found to be related to virus stimulation. However, the expression of the gene is controlled by a complex gene network and environmental factors including virus virulence, feeding conditions, stress and the like, which brings great interference and difficulty to the monitoring of the antibody level after the swine fever immunization. Therefore, only the complex immune mechanism is limited in the aspect of genetic research, scientists hopefully search the relevant gene of swine fever immunity through the research of a virus-organism-environment interaction mechanism, and stably distinguish the antibody level after the swine fever immunity through DNA methylation and molecular markers, thereby laying a theoretical foundation for epigenetic treatment and prevention of the swine fever.

The sequencing technology of DNA methylation mainly comprises Bisulfit sequencing, Bsulfit clone sequencing, pyrosequencing and the like. And analyzing DNA methylation differential sites by combining Bisulfit sequencing with clone sequencing, quickly and accurately quantifying one or more methylation sites in one detection through a pre-designed reaction system, and reacting the methylation state of individual cells by using Bisulfit sequencing results. The Bisulfit sequencing result is presented in a sequence form, the trend is consistent with the clone sequencing result, and the method is a research method for quickly and conveniently detecting the DNA methylation of the target gene at present. In addition, the technology can detect SNP mutation of methylation sites at the same time. In humans, more than 10% of the consensus SNPs for different alleles overlap with differentially methylated regions. Thus, differences in DNA methylation may be affected by genetic differences between animals. Because DNA methylation is cell-specific, particular attention should be paid to cell populations in studying immune responses, e.g., there is some difference in DNA methylation status between whole blood cells and lymphocytes. The use of the blood can reflect the immune status of the whole immune cells, and the research on the immunity of the pig body by using the whole blood in the livestock production is more convenient and reliable. The stable, reliable and simple operation Bisulfit sequencing method is utilized to carry out DNA methylation quantification of antibody level for distinguishing swine fever immunity, and the method has important significance for swine fever immunity research in production.

The porcine CD4Gene is located on chromosome 5 (Genebank accession No.: NC 010447; NCBI: Gene ID 404704), has a full length of about 26Kb (containing 11 exons and 10 introns), and encodes the leukocyte surface antigen CD4 protein. Leukocytes are a type of nucleated cells, and the leukocytes commonly found in normal porcine blood mainly include neutrophils, lymphocytes, and monocytes. The initial resistance encountered by the virus to invade the body is derived from the white blood cells, which are therefore the defense cells of the body. Lymphocytes, a cell with specific immune function, can be further divided into T lymphocytes and B lymphocytes, which perform cellular and humoral immune functions, respectively. Mature T lymphocytes are classified into CD4+ helper T cells (containing CD4 differentiation antigen) and CD8+ anti-toxic T cells (containing CD8 differentiation antigen) according to their surface antigen signature and function. The main functions of CD4+ T cells are to enhance and amplify the immune response process, activate macrophages, activate humoral immunity and promote humoral immunity through secreted lymphokines. The mature CD4+ T cell has CD4 differentiation antigen on the surface, and participates in immune response. Swine fever virus infected pigs show a dramatic change in the ratio of CD4+ T cells. Furthermore, the ratio of CD4+/CD8+ was much higher in uninfected herds than in infected herds. In recent years, epigenetic modifications of the CD4gene have been found to have a significant effect on T cell expression during immune processes. For example, epigenetic suppression of the CD4gene can affect the immune response. Our previous studies found that the CD4gene is an important candidate gene related to viral response, but no report is made on the influence of the DNA methylation level of the porcine CD4gene on the level of antibodies raised against swine fever.

Disclosure of Invention

The invention aims to provide a method and a primer pair for quantitatively detecting different levels of swine fever antibodies, and the influence of the DNA methylation level of a swine CD4gene on the level of swine fever immune antibodies can be researched by successfully marking DNA methylation molecules, so that the method and the primer pair have great significance; meanwhile, the method provided by the invention is convenient to use and high in sensitivity, and can be used for detecting the level of swine fever antibodies of white pigs and long-white pigs and identifying the immune effect. The invention provides a new method for detecting the level of the antibody of the swine fever, and has great application value and social benefit.

A primer pair for quantitatively detecting different levels of swine fever antibodies is prepared into a reagent for quantitatively detecting pigs with different swine fever antibody levels by the primer pair: the reagent comprises a primer pair consisting of DNA shown by SEQ ID N0.5 and DNA shown by SEQ ID N0.6, a primer pair consisting of DNA shown by SEQ ID N0.7 and DNA shown by SEQ ID N0.8, and a quantitative detection primer pair consisting of DNA shown by SEQ ID N0.9 and cDNA shown by SEQ ID N0.10.

The primer pair consisting of the DNA shown in SEQ ID N0.5 and the DNA shown in SEQ ID N0.6, the primer pair consisting of the DNA shown in SEQ ID N0.7 and the DNA shown in SEQ ID N0.8, and the quantitative detection primer pair consisting of the DNA shown in SEQ ID N0.9 and the cDNA shown in SEQ ID N0.10 can be used for preparing a kit for quantitatively detecting pigs with different swine fever antibody levels.

The primer pair consisting of the DNA shown in SEQ ID N0.5 and the DNA shown in SEQ ID N0.6, the primer pair consisting of the DNA shown in SEQ ID N0.7 and the DNA shown in SEQ ID N0.8, and the quantitative detection primer pair consisting of the DNA shown in SEQ ID N0.9 and the cDNA shown in SEQ ID N0.10 can be used for quantitatively detecting the DNA methylation levels of different swine fever antibodies.

A method for quantitatively detecting different levels of swine fever antibody, comprising the steps of: detecting the methylation degree of CpG (and Y) positions in DNA shown in a sequence 2 of a sequence table in a No. 5 chromosome of a pig to be detected, and determining whether the methylation level of the pig to be detected is hypermethylation or moderate methylation, wherein the swine fever antibody level of the pig with the hypomethylation level is lower than that of the pig with the hypermethylation level.

It will be clear to those skilled in the art that there are many assays available for detecting the presence of DNA methylation modifications in the promoter region sequence of the CD4 gene. These techniques include (but are not limited to): DNA sequencing, DNA chip, methylase specific PCR, PCR-RFLP, quantitative PCR, etc.;

the determination of whether the CD4gene promoter region of the peripheral blood of the pig to be detected is highly methylated or moderately methylated and the methylation degree specifically comprises the following steps:

(1) extracting the genome DNA of the pig blood and the lymphocyte to be detected;

(2) bisulfite treatment of genomic DNA;

(3) taking genome DNA as a template, and carrying out PCR amplification by using a primer pair consisting of DNA shown by SEQ ID N0.3 and DNA shown by SEQ ID N0.8 to obtain a PCR amplification product;

(4) performing hot start PCR amplification by using genomic DNA treated by bisulfite as a template and using a primer pair consisting of DNA shown by SEQ ID N0.2 and DNA shown by SEQ ID N0.6 in a sequence table to obtain a PCR amplification product;

(5) and (5) quantitatively detecting DNA methylation. The PCR amplification products were hot-started and subjected to sequencing by cloning using an ABI 377 automatic sequencer. The methylation degree of 9 CpG loci (1 st to 9 th CpG) can be quantitatively detected by SEQ ID N0.6, and the SNP genotype of the 2 nd CpG locus can be detected by SEQ ID N0.8;

(6) extracting RNA of the pig blood to be detected and carrying out reverse transcription to obtain cDNA;

(7) and (3) performing fluorescent quantitative PCR amplification by using a primer pair consisting of the cDNA shown in SEQ ID N0. column 4 and the cDNA shown in SEQ ID N0.10 by using the cDNA as a template to obtain the Ct value of a PCR product.

Preferably, the pigs are in particular big white pigs and long white pigs.

Preferably, the pig to be detected can be a pig with a high DNA methylation modified CD4gene and a pig with a medium DNA methylation modified CD4 gene.

Preferably, the method for detecting the level of swine fever antibody in the invention is a swine immunized with low level swine fever antibody of the moderate DNA methylation modified CD4gene identified by the above method.

Preferably, the DNA sequence shown in SEQ ID N0.2 is a bisulfite converted DNA sequence.

The DNA shown in SEQ ID N0.2 is used for preparing a kit for quantitatively detecting the antibody levels of different swine fever.

The invention has the beneficial effects that: aiming at 9 CpG loci in CD4gene promoter regions of peripheral blood and lymphocyte PBMCs in large white pigs and long white pigs in China, the invention discovers that the G/A SNP mutation of a second locus and the DNA methylation level thereof have obvious difference between the high swine fever antibody immunity level and the low swine fever antibody immunity level and obviously influence the gene expression (p is less than 0.01) for the first time, wherein the large white pigs with the low swine fever antibody level have obvious CD4 hypomethylation high expression and are expressed as GA heterozygotes. The method provided by the invention is convenient to use and high in sensitivity, and can be used for detecting the swine fever antibody level of white and long-white pigs and identifying the immune effect. The invention provides a new method for detecting the level of the antibody of the swine fever, and has great application value and social benefit.

Drawings

FIG. 1 shows partial pig DNA extraction

A, anticoagulant DNA extraction; extracting DNA of B lymphocyte PBMCs, and extracting anticoagulant RNA. M: DL2000 molecular weight markers; 1-7: test samples No. 1-7.

FIG. 2 PCR amplification Effect of porcine CD4Gene

A is used for repressing a 365bp PCR fragment of clone sequencing of sulfate; b224 bp PCR fragment for SNP sequencing; m: DL600 molecular weight markers; 1-7: test sample No. 1 to 7 8: and (5) negative control.

FIG. 3 shows the individual sequencing and SNP sequencing results of the bisulfite gene of the large white pig CD4 gene.

A peripheral blood CD4 methylation sequencing; CD4 methylation sequencing of B PBMCs; c peripheral blood CD4 SNP sequencing results; CD4 SNP sequencing results of DPBMCs.

FIG. 4 is a PCR electrophoresis identification chart of recombinant plasmid

Circle a represents randomly picked clones; b, PCR of recombinant plasmid.

FIG. 5 sequencing of methylated clones of GA heterozygotes from large white pigs.

A degree of DNA methylation after sequencing of peripheral blood CD 4; degree of DNA methylation after CD4 sequencing of B PBMCs.

FIG. 6 shows a comparison of the degree of methylation of CD4 DNA at different levels in the immunization of large white and long white swine fever antibodies.

A, methylation degree of a low swine fever antibody of a big white pig; b, methylation degree of the big white pig high swine fever antibody; c, methylation degree of low swine fever antibody of the Changbai pig; d, methylation degree of the Changbai pig high swine fever antibody.

FIG. 7 detection of peripheral blood CD4gene expression level under different antibody levels of hog cholera.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the following examples,% is by mass unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 optimized DNA extraction of blood and PBMCs

Anti-coagulation DNA extraction

Anticoagulant Genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega, Beijing agency) optimization method. And (3) uniformly mixing blood samples, taking 300 mu L of whole blood, adding 900 mu L of cell lysate, reversing and uniformly mixing, incubating at room temperature for 10min to lyse red blood cells, vortexing by a vortexer for 5-10 s at 14000rpm, centrifuging for 20s, removing supernatant, and leaving white precipitates (if the blood samples are frozen, repeating the steps until the precipitates become white). Vortex for 10-15 s to resuspend the white precipitate, add 300. mu.L of lysis solution to the resuspended cells, blow-blow the lysis with a pipette, make the solution viscous (if cell clumps are visible after mixing, incubate for 1h at 37 ℃), add 1.5. mu.L of RNase solution to the lysis solution to remove RNA contamination, incubate for 15min at 37 ℃, cool to room temperature. And detecting by 1% agarose gel electrophoresis.

Second, separation of porcine anticoagulant lymphocyte PBMCs

1ml of fresh blood (K3 EDTA anticoagulation), adding 1640 serum-free culture solution or 1 XPBS reagent at a ratio of 1: 1; a porcine lymphocyte separation medium (Ficoll medium, TBD) was added to a 15ml/50ml centrifuge tube. Centrifuge at room temperature, 2000rpm, using a flat bottom centrifuge for 25 min. The cells are divided into four layers which are respectively as follows from top to bottom: plasma and tissue homogenate liquid layer, annular milky white lymphocyte layer, transparent separation liquid layer, red blood cell layer) carefully taking out the white blood cell layer, re-suspending in 4ml of PBS, uniformly mixing, centrifuging at 1500rpm for 10min, repeating the previous step of washing for 2-3 times, and collecting cells for DNA extraction.

Third, extraction of PBMCs DNA of pig

PBMCs Genomic DNA was extracted by the Wizard Genomic DNA Purification Kit (Promega, Beijing agency) optimization method. Washing cells with 200 mu LPBS at 16000rpm, centrifuging for 10min, adding 600 mu L of nuclear lysate, repeatedly blowing and sucking until cell mass disappears, incubating at 37 ℃ for 15min with 1.5 mu LRNase enzyme, adding 200 mu L of protein precipitation solution, violently shaking for 20s, cooling on ice for 5min, 16000rpm, centrifuging for 4min, and forming white compact protein precipitate. Taking a clean 1.5ml centrifuge tube, adding 600 mu L of isopropanol, carefully taking supernatant, adding the isopropanol, reversing and uniformly mixing until white linear DNA forms precipitate, centrifuging at 16000rpm for 1min, removing supernatant, adding 600 mu L of 70% ethanol, reversing for several times to clean the DNA precipitate, centrifuging at 16000rpm for 1min, gently sucking away the ethanol (avoiding sucking away the precipitate), naturally drying for 10-15 min, dissolving the DNA by using 100 mu L of DNA hydration, and incubating for 1h at 65 ℃ or overnight at 4 ℃. And detecting by 1% agarose gel electrophoresis.

Example 2 optimization of PCR reaction

First, genome DNA bisulfite treatment

Quantitative DNA was treated with bisulfite using EZ DNA Methylation-Gold Kit (Zymo research, Beijing Tianmo scientific and technological agency) optimization method. Mixing 130 μ L CT Conversion Reagent and 20 μ L DNA (1 μ g) in a PCR tube, mixing, performing PCR thermal cycle, adding 600 μ L M-Binding buffer, mixing by inversion, centrifuging at 12000rpm for 30s, and discarding the waste solution; 100 mu L M-Wash buffer, loading into a column, centrifuging at 12000rpm for 30s, and discarding the waste liquid; 200 mu L M-depletion buffer, placing in a column, standing at room temperature for 30min, centrifuging at 12000rpm for 30s, and discarding the waste liquid; 200 mu L M-Wash buffer, loading into a column, centrifuging at 12000rpm for 30s, and discarding the waste liquid; 20 u L M-Elution buffer, put into a column, transferred into a new centrifuge tube, placed for 20min at room temperature, 12000rpm, centrifuged for 30s, collected the transformed DNA, diluted 1:5, stored at-20 ℃.

Hot start PCR based on bisulfite sequencing

The PCR 50. mu.L amplification system was: 2 × Reaction Buffer 25 μ L, dNTP Mix 0.25 μ L, upstream and downstream primers 1 μ L each (10 μmol/L), bisulfite treated DNA template 6 μ L, Zymo Taq enzyme 0.4 μ L (Zymo research, Kyoto Tianmo scientific agency), and DNase/RNase free water to 50 μ L. The reaction conditions are as follows: denaturation at 95 deg.C for 10 min; 30s at 94 ℃, 40s at 50-60 ℃, 45s at 72 ℃ and 40 times of circulation; extension at 72 ℃ for 7min and storage at 4 ℃.

PCR based on SNP sequencing

The PCR 50. mu.L amplification system was: 2 XTaq PCR Master Mix 25. mu.L (Beijing Zhongkotay, RTC 3104-03), upstream primer 1. mu.L (10. mu. mol/L), downstream primer 1. mu.L (10. mu. mol/L), genomic DNA template 4. mu.L, Dnase/Rnasefree water to 50. mu.L. The reaction conditions are as follows: denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 50-60 ℃, 45s at 72 ℃ and 35 times of circulation; extension at 72 deg.C for 5min, and storing at-20 deg.C in dark.

Fourth, detection of PCR amplification result

After amplification, 2. mu.L of PCR product was detected on 2% agarose gel, and a band with a stronger single signal was obtained without primer dimer or other non-specific bands.

Example 3 direct bisulfite sequencing

First, bisulfite mixed pool sequencing and individual direct sequencing

The method comprises the steps of (1) mixing the DNA treated by the bisulfite diluted at a ratio of 5 in an equivalent mixing tank, using the tank DNA as a template, carrying out temperature gradient hot start PCR amplification to obtain a PCR product at a proper temperature, and using ABI company 377 to directly carry out sequencing detection after the PCR product is qualified by gel electrophoresis detection. The individual sequencing is to directly sequence the individual DNA after the last step of temperature amplification (Beijing Yihui Yuan).

Cloning and sequencing bisulfite

(1) Cutting and recovering the amplified DNA

(2) Connection of

Adding the recovered target fragment and the pGM-T vector into a reaction tube, uniformly mixing, and placing the reaction tube and the pGM-T vector in a 15 ℃ water bath kettle for overnight chaining;

(3) transformation of recombinant plasmids

Under a completely sterile state, a tube of 100. mu.L of fresh DH 5. alpha. E.coli competent bacteria was taken out of a-80 ℃ freezer and hand-thawed; adding 5 μ L of the linked product into each competent cell, mixing gently, and standing on ice for 30 min; thermally activated for 90s in water bath at 42 ℃; rapidly transferring EP tube to ice, and rapidly cooling cells for 1-2 min; in the sterile operating station, 600. mu.L of LB liquid medium (no AMP) was added to the EP tube and the bacteria were revived (200 rpm, 50min, 37 ℃); 100 μ L of resuscitating bacteria was plated on LA plate medium. Culturing overnight in an incubator at 37 ℃; the next day, the growth of the colonies in the culture dish was observed, and 8-10 colonies were randomly picked and streaked on another LA solid medium (8-12 h).

(4) Identification of recombinant plasmids

Performing PCR on thalli, wherein the original PCR amplification reaction is the same, and only the DNA in the thalli is changed into bacterial liquid;

after PCR, the PCR products were sent to Beijing Tianyihuiyuan company for sequencing.

Example 4 porcine anticoagulant RNA expression assay

Anti-coagulation RNA extraction

Mixing fresh anticoagulated blood and cell lysate (RL) at a ratio of 1:3, and fully lysing; 0.2ml of chloroform was added to 1ml of RL, shaken vigorously for 15s and incubated at room temperature for 3 min. Centrifuging at 4 ℃ and 12000rpm for 10min, and dividing the sample into 3 layers; taking the upper aqueous phase, wherein the volume of the upper aqueous phase is 60% of RL, namely taking 0.6ml to put into a new centrifugal tube, adding 70% ethanol with the volume being 1 time of RL, and reversing and mixing evenly, wherein precipitates can appear; the solution and the precipitate were transferred to an adsorption column (RP 1202, Baitach Biotechnology Co., Ltd., Beijing) at 10000rpm, centrifuged for 45 seconds, and the waste liquid was discarded. Adding 50 μ L deproteinized solution RE (hectarex), 12000rpm, centrifuging for 45s, and discarding waste liquid; adding 700 μ L rinsing solution RW (DEPC water with 70% alcohol), 12000rpm, centrifuging for 60s, and discarding the waste liquid; adding 500 μ L of rinsing solution RW at 12000rpm, centrifuging for 60s, and discarding the waste liquid; 12000rpm, centrifuging for 2min, idling; the adsorption column was put into an RNase-free centrifuge tube, 30. mu.L RNase-free water was added, incubated at 65 ℃ for 10min, allowed to stand at room temperature for 2min, and centrifuged at 12000rpm for 1min to collect blood RNA.

II, anticoagulation RNA reverse transcription cDNA

Oligo (dT) was used to initiate first strand cDNA synthesis. Initial template amount: total RNA 2. mu.g, 1. mu.L control RNA (50 ng/. mu.l). Before use, each component in the kit is shaken up and centrifuged briefly; the following components were added to a 0.2ml sterile PCR tube: prepare RNA/primer mix as in table 1:

TABLE 1 RNA/primer mix reactions

Incubating the mixture at 65 deg.C for 5min, and standing on ice for at least 1 min; another sterile PCR tube was prepared with 2 × reaction mix as shown in table 2:

TABLE 22 Xreaction mix reaction

Adding 9 μ l of 2 × reaction mixture into the mixture of the first step, gently mixing, centrifuging for a short time, and incubating for 2min at 42 ℃; add 1. mu.l GoldScript RT to each tube, add 1. mu.l DEPC water without RT control, incubate for 50min at 42 ℃; incubating at 70 deg.C for 15min, terminating the reaction, and cooling on ice; carrying out short-time centrifugal collection reaction; add 1. mu.l RNaseH to each tube and incubate at 37 ℃ for 20 min; storing at-20 ℃ or performing fluorescent quantitative PCR reaction. cDNA was diluted 1:50 fold before fluorescent quantitation PCR.

Third, reverse transcription PCR of cDNA

The PCR 50. mu.L amplification system was: 2 XTaq PCR Master Mix 25. mu.L (Beijing Zhongkotay, RTC 3104-03), upstream primer 1. mu.L (10. mu. mol/L), downstream primer 1. mu.L (10. mu. mol/L), genomic DNA template 4. mu.L, Dnase/Rnasefree water to 50. mu.L. The reaction conditions are as follows: denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 60 ℃, 45s at 72 ℃ and 35 times of circulation; extension at 72 deg.C for 5min, and storing at-20 deg.C in dark.

Design of CD4 Real time PCR primer and Real time PCR reaction condition

The primers of the target gene and the housekeeping gene were designed by using Oligo6.0 software, and the sequences were synthesized by Shanghai Invitrogen, and the sequences of the primers are shown in Table 3. The same procedure was used for each q-PCR reaction: incubate at 95 ℃ for 10min, amplify 45 cycles (95 ℃ 10s, 60 ℃ 10s, and 72 ℃ 10 s), and the dissolution profile is reduced using high temperature and cooling. The crossover point (Cp value) was determined using the second derivative maximum analysis tool in Roche 480 software. The RT-PCR technology is combined with the 2-delta-Delta Ct method to calculate the relative expression quantity of the CD4 gene. The reaction conditions are shown in Table 3:

TABLE 3 Real time PCR reaction System

Example 5 Swine fever antibody

Horizontal detection of DNA methylation of CD4

Gel electrophoresis detection for extracting DNA (deoxyribonucleic acid) from porcine K3EDTA (ethylene diamine tetraacetic acid) anticoagulation

As can be seen from FIG. 1A, 10-20 μ g of DNA was extracted from anticoagulated blood, and the OD A260/A280 values detected by a spectrophotometer were all between 1.80-2.0, and the OD A260/A230 values were all between 1.50-1.8, indicating that the protein removal effect was good, and there were no residual organic solvents such as RNA and ethanol. Provides a basis for the subsequent study of DNA methylation. It can be seen from FIG. 1B that the DNA concentration extracted from the lymphocyte PBMCs is high and there is no foreign RNA contamination.

As can be seen from FIG. 1C, the anticoagulant RNA extraction concentration is qualified at 200-300 ng/. mu.L, the OD A260/A280 values are all between 1.80-2.0, and the OD A260/A230 values are all between 1.30-1.5, indicating that there are no residual organic solvents such as DNA and ethanol. RNA is not degraded basically, and a foundation is provided for the subsequent research of RNA expression.

Second, detection of agarose gel of PCR product of porcine CD4gene for bisulfite sequencing and clone sequencing

The CD4gene shown in FIG. 2 is used for amplification results of bisulfite sequencing and pyrophosphate sequencing, and has bright and regular electrophoresis bands, no non-specific bands, no primer dimer and no pollution, which indicates that the amplification effect is better and methylation sequencing analysis can be carried out. The concentration of PCR products used for methylation sequencing was lower than the PCR products used for SNP sequencing under 40 cycles of amplification conditions.

Third, sequencing individual bisulfite of porcine CD4gene and SNP sequencing result

In order to preliminarily detect whether the CpG loci of blood and lymphocytes are methylated, the invention firstly carries out hybridization sequencing on PCR products. FIG. 3 shows the results of 1 individual bisulfite sequencing (FIGS. 3A and 3B) of the blood and lymphocyte porcine CD4gene and SNP sequencing (FIGS. 3C and 3D) of the same locus, which result in 9 total CpG loci. FIGS. 3A, 3B show 2 CpG sequenced loci with red and blue doublets indicated as differentially methylated loci. Black arrows indicate the degree of methylation of the first CpG locus, averaging 58.1% (peripheral blood) and 79.8% (lymphocytes); the red arrow indicates the SNP locus, and the green-black double peak indicates heterozygotes for the G/A mutation. The red color of FIGS. 3C, 3D shows that the individual was identified as heterozygous based on genomic DNA sequencing.

Fourthly, selection of clone and PCR result of recombinant plasmid

The purified PCR product was cloned and transformed with recombinant plasmid, and then subjected to PCR electrophoresis detection, and the positive band was picked up, with the result shown in FIG. 4. FIGS. 4A and 4B are black circles representing 8 clones obtained by randomly selecting peripheral blood and cloning PBMCs CD4gene for recombinant plasmid transformation and sequencing; FIG. 4C represents the PCR electrophoretic identification of recombinant plasmids, wherein 7 of 1, 3, 7, 8, 9, 10 and 11 are the target bands for the subsequent clone sequencing analysis.

Methylation clone sequencing of pig GA heterozygote

The cloning and sequencing of bisulfite PCR products is the traditional detection method of DNA methylation, and we further adopt the method to judge whether the CpG loci are indeed modified by DNA methylation. FIG. 5 shows the sequencing results of bisulfite clone of CD4gene of porcine peripheral blood and PBMCS, and the average DNA methylation degree of 9 CpG loci in two cells of 9 YG loci were 91.7% and 94.4%, respectively. The results show that the mean level of differential methylation loci of the CD4gene promoter regions of the two cells is relatively consistent. However, there were more significant differences at the second CpG locus (indicated by the red arrows in FIG. 5, averaging 15% and 31.5%). Peripheral blood whole blood cells will therefore be selected for analysis of the degree of methylation at the second differentially methylated locus.

Comparison of different levels of CD4 DNA methylation degree of hog cholera antibody of six, large and long white pigs

In order to analyze the methylation difference and SNP difference of CD4gene promoters of large white pigs and long white pigs under different swine fever antibody levels, 5 GA heterozygous individuals of the large white pigs and 5 homozygous individuals of the GG of the long white pigs are selected for the experiment to analyze the methylation difference of CD 4. The high antibody group, CD4, was observed to be methylated to a higher degree in both varieties than the low antibody group. The methylation degree of the Changbai pig CD4gene is generally higher than that of the Dabai pig. In the high antibody group, the methylation degree of the long white pigs at the CpG locus (97.3%) is about 20% higher than that of the large white pigs (72.4%). In the low antibody group, the methylation level of CD4 (82.1%) in the same locus of long white pigs was higher (67.9%) than that of large white pigs (fig. 6), and the differential methylation level was about 15%.

Seventhly, detecting peripheral blood CD4gene expression level under different levels of hog cholera antibody

We continued to analyze the methylation degree of CD4 DNA in peripheral blood of the large white pig and the long white pig, and found that the methylation degree of the long white pig at a CpG locus is slightly higher than 20% of that of the large white pig, and the mutation of G/A is found at the YG locus of the long white pig breed, which may be one of the important reasons for the down-regulation of the CD4gene expression. Further proves the DNA methylation regulation mechanism of the weakened resistance of the long and white pigs before and after the immunization of the swine fever attenuated vaccine. FIG. 7 shows the difference of CD4 expression levels of different antibodies of white and long pigs immunized with hog cholera attenuated vaccine. The results show that the expression level of CD4 is reduced in the large white pig high antibody group, and the difference is very significant from the low antibody group, P = 1.5402E-07 (fig. 8). In the high antibody group, the degree of DNA methylation of the GG-type large white pig was increased by about 15% compared with the low antibody group. It was preliminarily shown that the CD4 promoter and SNP affected the expression of the CD4 gene. The hog cholera attenuated virus may cause the self-immune suppression of the body, cause the transient suppression of the expression of the CD4gene, and further cause the functional inactivation of CD4+ T cells.

Sequence listing

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<213> sequence After bisulfite conversion of CD4Gene (After the After bisulfate transformation of CD4gene)

<400>2

tatggaggtt tttaggttag gggtttgatt ggagttatag atgttggttt atattagagt 60

tatagtaaag ttagatttga gttgygtttt taatttgtat yggtttattg taaygtygga 120

tttttaattt attgagygag ggtagggaty gaattygaaa ttttatggtt tttaggtgga 180

ttygttaatt attgagttat aaygggaatt tttaaaaaaa aattttttaa ggggtttgtt 240

tagtttttaa ttttaattat gaatataagt gtttttgttg aggtaattat tatattttat 300

ttgtagttaa agtgttttat gttatttttt attttataat agagtgtatt taagagttga 360

g 361

<210>3

<211>224

<212>DNA

<213> SNP sequencing sequence (SNP sequencing sequence)

<400>3

cttacaccag agccacagca aagccagatc tgagctgcgt cttcaacctg caccgcggtt 60

cactgcaacg ccggatcctt aacccactga gcgagggcag ggatcgaacc cgaaacctca 120

tggttcctag gtggattcgt taatcactga gccacaacgg gaactcccaa aaaaaatttt 180

tttaaggggt ttgttcagct cctaactcca actatgaaca caag 224

Claims (8)

1. A primer pair for quantitatively detecting different levels of swine fever antibodies is characterized by comprising the following primer pairs:

5’-TATGGAGGTTTTTAGGTTAGG-3’

5’-CTCAACTCTTAAATACACTCT-3’

5’-CTTACACCAGAGCCACAGC-3’

5’-CTTGTGTTCATAGTTGGAGTT-3’

5’-CCTTCTGTTTCTCGCTGGG-3’

5’-GGCAGGTCTTCTTCTCACTA-3’。

2. the primer pair for quantitatively detecting different levels of swine fever antibodies according to claim 1, wherein: the primer pair is utilized to prepare a kit for quantitatively detecting pigs with different levels of swine fever antibodies.

3. A primer pair for quantitatively detecting different levels of swine fever antibodies is characterized in that: the primer pair can be used for quantitatively detecting pigs with different swine fever antibody levels.

4. A method for quantitatively detecting different levels of swine fever antibodies, characterized by comprising the steps of: detecting the methylation degree of CpG and Y positions in DNA shown by SEQ ID N0.2 in the chromosome 5 of the pig to be detected, and determining whether the methylation level of the pig to be detected is hypermethylation or moderate methylation, wherein the swine fever antibody level of the pig with the hypomethylation level is lower than that of the pig with the hypermethylation level.

5. The method of claim 4, wherein the antibody is selected from the group consisting of: determining peripheral blood of pigs to be testedCD4Whether the gene promoter region is highly methylated or moderately methylated and the methylation degree can specifically comprise the following steps:

(1) extracting the genome DNA of the pig blood and the lymphocyte to be detected;

(2) bisulfite treatment of genomic DNA;

(3) taking genome DNA as a template, and carrying out PCR amplification by using a primer pair consisting of DNA shown by SEQ ID N0.3 and DNA shown by SEQ ID N0.8 to obtain a PCR amplification product;

(4) performing hot start PCR amplification by using genomic DNA treated by bisulfite as a template and using a primer pair consisting of DNA shown by SEQ ID N0.2 and DNA shown by SEQ ID N0.6 to obtain a PCR amplification product;

(5) DNA methylation quantitative detection; carrying out clone sequencing on the PCR amplification product by using an ABI 377 automatic sequencer, wherein the methylation degree of 9 CpG loci can be quantitatively detected by SEQ ID N0.6, and the SNP genotype of the 2 nd CpG locus can be detected by SEQ ID N0.8;

(6) extracting RNA of the pig blood to be detected and carrying out reverse transcription to obtain cDNA;

(7) and (3) performing fluorescent quantitative PCR amplification by using a primer pair consisting of the cDNA shown in SEQ ID N0.4 and the cDNA shown in SEQ ID N0.10 by using the cDNA as a template to obtain the Ct value of a PCR product.

6. The method of claim 4 or 5, wherein the antibody is used for quantitative determination of different levels of swine fever antibody: the pig is especially big white pig and Changbai pig.

7. The method of claim 4, wherein the antibody is selected from the group consisting of: the pig with the moderately DNA-methylation modified CD4gene identified by the method described above was used for differentiation.

8. The method of claim 4, wherein the antibody is selected from the group consisting of: the degree of methylation of the 1 st to 9 th CpG loci can be quantitatively detected by SEQ ID N0.6.

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