CN103966312B - Construction of PD-L2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, real-time detection method of gene abundance and its application - Google Patents

Abstract

The invention belongs to molecular pathology and immunological technique field, relate to the structure of pig peripheral blood monokaryon lymphocyte PD-L2 recombinant plasmid, gene abundance real-time detection method and application thereof.By gathering the lymphocytic total serum IgE of pig peripheral blood monokaryon, total serum IgE reverse transcription is become cDNA; Adopt regular-PCR amplification PD-L2 goal gene fragment, detect through agarose gel electrophoresis, and reclaim purifying; By PD-L2 goal gene fragment and pMD? 18-T carrier connects, and is converted in competent cell DH5 α, extracts recombinant plasmid; After colony screening, carry out sequencing analysis, to choose with the positive plasmid of goal gene fragment identical sequence as standard substance plasmid, be depicted as typical curve by copy concentrations; The gene abundance of PD-L2 is measured according to fluorescent signal change and typical curve.Pig PD-L2 gene abundance real-time detection method of the present invention have detect that flux is high, susceptibility is high, high specificity, easy and simple to handle, cost is low and the advantage such as quantitatively accurate.

Description

Construction of PD-L2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, real-time detection method of gene abundance and its application

technical field

The invention belongs to the technical field of molecular pathology and immunology, and specifically relates to the construction of a PD-L2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, a real-time detection method for gene abundance and an application thereof.

Background technique

Classical swine fever (CSF) is an acute, febrile, highly contagious infectious disease caused by classical swine fever virus (CSFV). Studies have shown that CSFV mainly infects the monocyte-macrophage system, causing severe lymphopenia, platelet aggregation and coagulation dysfunction, and atrophy of central immune organs such as thymus and bone marrow. CSFV can also cause immune function damage by reducing the body's antiviral response, inhibiting the apoptosis of infected cells, and promoting the apoptosis of uninfected cells.

Programmed death ligand (PD-L2) and its programmed death receptor-1 (PD-L2) are members of the CD28/B7 superfamily and are widely expressed in various tissues. PD-L2/PD-L co-stimulatory signals are activated or inhibited depending on the degree of inflammation, immune status and genetic background of the body, and are mainly involved in the central and peripheral immune tolerance of T cells. Compared with other members of the CD28 superfamily, the PD-L2/PD-L signaling pathway plays a more extensive and complex role in immune regulation, and is closely related to autoimmune diseases, tumors, chronic infection and inflammation. In terms of immune regulation, PD-L2 and its ligand PD-Ls jointly mediate the activation of the PD-L2:PD-L signaling pathway, transmit immune tolerance and immunosuppressive signals in the central immune and peripheral immune responses, and are specific to antigens The function of T and B cells, the proliferation of T cells, the secretion of cytokines and the killing ability all have an inhibitory effect. After blocking this pathway, most of the functions of T cells can be restored. It has been confirmed by related studies that the infection of some viruses can induce the expression of PD-L2 and its receptor PD-Ls, thereby affecting the PD-L2:PD-Ls pathway, making the virus escape the surveillance and killing of the immune system, and causing the body to produce immunosuppression and persistent infection. Therefore, PD-L2 and its ligand PD-Ls have gradually become the focus of attention in the research of chronic persistent viral infection, immune tolerance disease, tumor and other immunosuppressive diseases in recent years. Or in terms of persistent viral infection, the research on the T cell immunosuppressive pathway PD-L2:PD-Ls has just started, especially the research on porcine viral diseases is still rarely reported.

In recent years, real-time fluorescent quantitative PCR (Real-TimePCR) technology has provided a new method for gene abundance detection. Compared with traditional methods, it has high sensitivity, strong specificity, easy operation, low cost and accurate quantification. application, it can achieve the purpose of real-time monitoring by detecting the fluorescent signal intensity of PCR amplification of the target sequence. However, the establishment and application of the Real-TimePCR detection system for the abundance of PD-L2 gene in porcine peripheral blood mononuclear cells has not been reported.

Contents of the invention

The technical problem to be solved by the present invention is: the present invention provides a method for constructing a positive standard recombinant plasmid of PD-L2 real-time fluorescent quantitative PCR in porcine peripheral blood mononuclear lymphocytes;

On this basis, the present invention establishes a real-time fluorescence quantitative PCR detection method for PD-L2 gene abundance, which has the advantages of high sensitivity and strong specificity;

The invention provides a technical platform for the quantitative analysis of porcine PD-L2 molecular detection by studying the change rule of PD-L2 in peripheral blood mononuclear cells, and can be used to analyze the expression of PD-L2 gene after porcine infection with viral diseases such as CSF Changes in transcription levels.

Technical scheme of the present invention:

A method for constructing a positive standard recombinant plasmid for PD-L2 real-time fluorescent quantitative PCR of porcine peripheral blood mononuclear lymphocytes, comprising the following steps:

Collect the peripheral blood of piglets, isolate the peripheral blood mononuclear lymphocytes, extract the total RNA of the peripheral blood mononuclear lymphocytes, reverse transcribe the total RNA into cDNA, store the cDNA at -20°C, and amplify PD by common PCR method - L2 target gene fragment;

Amplify the PD-L2 target gene fragment by common PCR method, detect the target gene fragment by agarose gel electrophoresis, recover and purify; then connect the purified PD-L2 target gene fragment with the pMD18-T vector, and transform it into a competent state Extract the recombinant plasmid from DH5α cells, perform sequencing analysis after cloning and screening, and obtain a positive standard recombinant plasmid with the same sequence as the target gene fragment;

The reaction system for ordinary PCR amplification is 25 μL, and the reaction system is as follows: 2 μL sample cDNA template, 2.5 μL 10×PCRBuffer, 2 μL dNTP, 0.5 μL each of the forward primer and reverse primer at a concentration of 20 μmol/L, and 0.5 μL of TaqDNA Polymerase, the rest is sterile triple-distilled water; common PCR reaction procedure: hot start at 95°C for 1min; denaturation at 94°C for 30s; annealing at 55°C for 30s; extension at 72°C for 30s, 30 cycles, and finally extension at 72°C for 10 minutes;

Among them, the forward primer F is: 5'-GCCAACACCAGCTATACCAAGAC-3'

The reverse primer R is: 5'-GGGATGAAAACACGGAGCA-3'.

The target gene fragment is SEQIDNo.1; the reaction procedure for ordinary PCR amplification: hot start at 95°C for 1min; denaturation at 94°C for 30s; annealing at 55°C for 30s; extension at 72°C for 30s, 30 cycles, and finally extension at 72°C 10min.

A method for real-time detection of the abundance of PD-L2 gene in porcine peripheral blood mononuclear lymphocytes, comprising the following steps:

(1) Ligate the recovered and purified PD-L2 target gene fragment with the pMD18-T vector, then transform it into competent cells DH5α, and extract the recombinant plasmid; perform sequencing analysis after clone screening, and select the same PD-L2 target gene fragment The positive plasmid of the sequence is used as the standard plasmid, the DNA concentration of the standard plasmid is measured, and converted into the copy concentration of the plasmid, and the positive plasmid is diluted in a concentration gradient of 1:10 times;

(2) Use the diluted positive plasmid as a template to carry out real-time fluorescent quantitative PCR amplification reaction, detect the fluorescent signal with a real-time fluorescent quantitative PCR instrument, draw a standard curve after the reaction, and then measure the sample according to the change of the fluorescent signal and the standard curve Gene abundance of PD-L2 in DNA;

The reaction system for real-time fluorescence quantitative PCR amplification is 20 μL, including 2 μL plasmid template, 0.2 μL each of forward primer F and reverse primer R at a concentration of 20 μmol/L, 10 μL of SYBRGreenIPreMix, and 7.6 μL of triple-distilled water;

Wherein, the forward primer F is: 5'-GCCAACACCAGCTATACCAAGAC-3';

The reverse primer R is: 5'-GGGATGAAAACACGGAGCA-3'.

The target gene fragment is SEQIDNo.1; the reaction program of the fluorescent quantitative PCR amplification is: 95°C hot start for 30s; 94°C denaturation for 5s; cycle.

The concentration range of the diluted positive plasmid is 2.66×10 ¹⁰ -2.66×10 ¹ copies/μL; the standard curve is Y=-3.334X+22.87.

The application of the real-time detection method for the abundance of PD-L2 gene in porcine peripheral blood mononuclear lymphocytes is used in the analysis of the variation law of the transcription level of PD-L2 gene after pigs are infected with viral diseases.

Positive beneficial effect of the present invention:

The present invention establishes a real-time fluorescence quantitative PCR method for PD-L2 gene abundance in porcine peripheral blood mononuclear cells by synthesizing forward primers, reverse primers, and PD-L2 target gene fragments, in order to better detect porcine viral Accurately quantify the transcription level of PD-L2 in the disease, and then study the change rule of PD-L2 in peripheral blood mononuclear cells, provide a technical platform for the quantitative analysis of porcine PD-L2 molecular detection, and provide a basis for PD-L2 in porcine virus It lays the foundation for the biological functions and mechanisms of their application in diseases.

Compared with the traditional technology, the technical solution disclosed in the present invention has higher sensitivity. Using the established method to detect 9 groups of standard positive plasmids with different concentrations of 10 ⁹ to 10 ¹ copies/μL, the results show that: PD in the present invention The lower detection limit of -L2 is 10copies/μL, and there is a good linear relationship between its concentration and C _t value, the correlation coefficient R ² =0.998; the repeatability test was carried out using the established method, and the repeatability test results within and between groups It shows that the coefficients of variation are all within 3%, with good repeatability; from the dissolution curve, it can be seen that a single narrow peak appears in the dissolution curve of PD-L2 molecule (Figure 4), and agarose gel electrophoresis analysis shows that Real-timeRT -The result of PCR amplification is a single specific target fragment (Figure 5), indicating that the present invention has good specificity; in addition, the technical solution has the characteristics of high detection throughput, simple operation, low cost and accurate quantification.

The present invention proves through experiments that after pigs are infected with CSFV, the expression level of PD-L2 gradually increases as the virus replicates in the body, indicating that the infection of CSFV and the viral load are closely related to the change of pig PD-L2 gene abundance; It was also confirmed that there is a certain relationship between the expression changes of PD-L2 and the expression changes of IL-2 and IL-10 cytokines after pigs infected with CSFV. , causing a decrease in the immune function of pig T cells, resulting in a decrease in the immune function of infected pigs.

The porcine PD-L2 gene real-time fluorescent quantitative PCR detection method of the present invention provides a technical platform for detecting the expression change law of PD-L2 in immune function damage caused by porcine viral diseases such as CSF (swine fever), thereby further enriching The mechanism of immune function damage caused by classical swine fever.

Description of drawings

Figure 1 is the result of agarose gel detection of porcine PD-L2 gene amplification;

Figure 2 is the amplification curve of real-time fluorescence quantitative analysis of porcine PD-L2;

Figure 3 is the standard curve for real-time fluorescence quantitative analysis of porcine PD-L2;

Figure 4 is the dissolution curve of the standard substance for real-time fluorescence quantitative analysis of porcine PD-L2;

Figure 5 shows the results of real-time fluorescent quantitative product specificity analysis of porcine PD-L2;

Figure 6 is the copy number of PD-L2 gene in the peripheral blood mononuclear cells of pigs infected with CSFV at different stages;

Fig. 7 is the detection result of CSFV viral load in the blood plasma of different periods of pig infection CSFV;

Fig. 8 is the copy number of IL-2 gene in different stages of pig infection CSFV;

Fig. 9 is the copy number of IL-10 gene in pigs infected with CSFV at different stages.

detailed description

In the present invention, gene copy number and gene abundance represent the same concept; Real-timePCR and real-time fluorescent quantitative PCR represent the same concept; ddH ₂ O represents double distilled water; CSF represents swine fever; CSFV represents swine fever virus, carboxyl Fluorescein diacetate succinimidyl ester (carboxyfluoresceinsuccinimidylaminoester, CFSE) is a fluorescent dye that can label living cells.

Experimental animals and bacterial strains in the examples: 30-day-old healthy piglets were raised in isolators; Shimen strain CSFV was provided by the China Veterinary Drug Inspection Institute, and the half infection rate was 10 ⁵ ×TCID ₅₀ ; Escherichia coli competent cells DH5α were Saved by this laboratory.

Main reagents and instruments: lymphocyte separation medium, purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.; PCR product purification and recovery kit, purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.; TaqDNA polymerase, reverse transcription kit, The pMD18-T vector was purchased from Dalian Bao Biological Engineering Co., Ltd.; SYBRGreenI was purchased from Invitrogen.

Example 1 Construction of porcine PD-L2 real-time fluorescence quantitative PCR positive standard recombinant plasmid

The peripheral blood of 45-day-old piglets was collected, and the lymphocytes in the peripheral blood were separated according to the instructions of the lymphocyte separation medium, and the total RNA was extracted according to the instructions of the Invitrogen TRIzol kit and related literature. The extracted total RNA was reverse-transcribed into cDNA according to the instructions of the reverse transcription kit, and stored at -20°C for use as a template for amplifying the PD-L2 target gene fragment.

Amplify the PD-L2 target gene fragment by common PCR method, detect the target gene fragment by agarose gel electrophoresis, recover and purify; then connect the purified PD-L2 target gene fragment with the pMD18-T vector, and transform it into a competent state Extract the recombinant plasmid from DH5α cells, perform sequencing analysis after cloning and screening, and obtain a positive standard recombinant plasmid with the same sequence as the target gene fragment;

The target gene fragment is the sequence SEQIDNo.1:

GCCAACACCAGCTATACCAAGACATCTGAAGGCCTCTACCAGGTCACTAGTGTTCTGCGCCTAAAGCCACACCCTGGTAGAAATTTCAGCTGTGTGTTCTGGAATGCCAACATGAAGGAATTTACTTTAGTCATCATCGTCCCAGGTGATATGGAAACCCACAAGACCCCTGCATCTTCACTGCTCCGTGTTTTCATCCC.

Common PCR reaction system for constructing PD-L2 real-time fluorescent quantitative PCR positive standard recombinant plasmid:

The general PCR reaction system is 25 μL, and the reaction system is as follows: 2 μL sample DNA template, negative control with sterile three-distilled water as template, 2.5 μL 10×PCRBuffer, 2 μL dNTP, 0.5 μL each of forward and reverse primers (20 μmol/L), 0.5 μL TaqDNA Polymerase (Dalian Bao Biological Engineering Co., Ltd.), and the remaining volume was made up with sterile three-distilled water; the reaction system was gently mixed, and the amplification reaction was carried out in an ordinary PCR machine.

The general PCR program is: hot start at 95°C for 1 min; denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s, 30 cycles, and finally extension at 72°C for 10 min.

Among them, the porcine PD-L2 real-time fluorescent quantitative PCR primers were synthesized by a biological company, and the primer sequences are as follows:

The forward primer F is: 5′-GCCAACACCAGCTATACCAAGAC-3′,

The reverse primer R is: 5'-GGGATGAAAACACGGAGCA-3'.

The normal PCR product was subjected to 2% agarose electrophoresis analysis, the voltage was 100V, and the product was observed under the ultraviolet light after 35 minutes. The result showed that a single band was amplified, and the fragment size was about 200bp.

The amplification results are shown in Figure 1. In the figure, M is the DL2000 molecular weight standard, and 1 is the amplification product of the PD-L2 gene. The amplification band is relatively clear, and there is no influence of primer-dimer. After the amplified sequence was cloned and sequenced, the nucleic acid homology comparison was carried out in GenBank. The results showed that the sequence homology of the obtained sequence SEQIDNo.1 and the porcine PD-L2 gene (NM_001204379) in GenBank was 100%. It shows that the primers and amplification products are correct and can be used in the next step of experimental research. After the specific fragments were recovered from the agarose gel, clone transformation was carried out, and the obtained positive clones were sent to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for sequencing analysis.

Example 2: Establishment of porcine PD-L2 real-time fluorescent quantitative PCR system

(1) Preparation of plasmid DNA template

The target gene fragment of PD-L2 was amplified by ordinary PCR amplification method, and the target gene fragment was detected by 2% agarose gel electrophoresis, recovered and purified, and then transformed into competent cells DH5α after ligation with pMD18-T vector (TaKaRa, Dalian) In , the recombinant plasmid was extracted; after clone screening, sequencing analysis was performed, and the sequencing results showed that it was 100% homologous to the sequence of the porcine PD-L2 gene in GenBank, indicating that the obtained sequence was correct, and the positive plasmid could be used for the production of plasmid standards. The standard plasmid DNA concentration was determined to be 84.7 μg/mL using a micronucleic acid protein spectrophotometer, and the initial standard plasmid solution was diluted 1:10 times.

(2) Calculation of standard plasmid concentration and creation of PD-L2 standard curve

Extract the plasmids of positive clones verified by sequencing, and convert the DNA concentration of the plasmids into copy numbers. The calculation method of plasmid copy number is: copy number (copies/μL)=concentration (μg/μL)×6.02×10 ²³ (copies/μL)/MW (g/mol).

Where MW = (cloning vector length + target fragment length) (bp)) × 660g/mol/bp.

Known: cDNA=84.7μg/mL, plasmid PMD18-T sequence length is 2692bp, PD-L2 gene insert fragment length is 200bp; the average molar mass of a single base is 660g/mol, the calculation formula is:

Molecular weight (MW)=(2692+200)×660=1.91×10 ⁶ g/mol.

Plasmid copy concentration: 6.02×10 ²³ ×(84.7×10 ^-9 ) ÷ (1.91×10 ⁶ )=2.66×10 ¹⁰ copies/μL.

The above-mentioned plasmid solution with known copy concentration was used as the standard plasmid, and was diluted in a 10-fold gradient (concentration range: 2.66×10 ¹⁰ -2.66×10 ¹ copies/μL) when drawing the Real-timePCR standard curve.

The plasmid with known copy number was serially diluted 1:10 times to obtain the plasmid standard.

Using the diluted plasmid as a template, real-time fluorescence quantitative PCR amplification reaction was carried out. The reaction was carried out using Real-timePCR kit SYBRPremixExTaqTM (Invitrogen Company). The reaction system was 20 μL: 2 μL standard plasmid template, F and R primers (20 μmol/L) each 0.2 μL, SYBRGreenIPreMix 10 μL, triple distilled water 7.6 μL. A negative control (template 2 μL ddH ₂ O) was set in each Real-time PCR reaction. The Real-time PCR program was: 95°C hot start for 30s; 94°C denaturation for 5s; 60°C annealing and extension for 34s; collect fluorescence signals at 60°C 40 loops. The F and R primers are the same as in Example 1.

Three parallel reactions were set up for the standard curve and all samples to be tested. The reaction was carried out in ABI7500 quantitative PCR instrument (LifeTechnologies, USA), and ABI7500 software was used to detect and collect fluorescent signals, and perform data analysis.

The real-time fluorescence quantitative analysis software of ABI7500 software draws the amplification curve of the reaction by itself (see Figure 2). The amplification curve shows that the six curves from left to right represent the amplification curves of the gradient dilutions of standard products 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , and 10 ² . Smooth, showing a typical S-type, and each cycle threshold (Ct value) is evenly spaced.

According to the concentration gradient of the standard provided by the amplification curve and the cycle threshold (Ct value) of the reaction, the ABI7500 software draws the standard curve of the reaction by itself (see Figure 3). The correlation coefficient of the standard curve R ² =0.998, the slope is -3.334, the standard curve is Y=-3.334X+22.87, where Y is the Ct value of the real-time fluorescent quantitative PCR reaction, and X is the standard plasmid copy constructed by the PD-L2 gene logarithmic value. The calculated amplification efficiency Eff%=100.618%, which meets the requirements of ABI7500 fluorescence quantitative analysis for the standard curve.

Measure the melting temperature of the standard substance of each concentration dilution gradient during the real-time fluorescent quantitative PCR process, and draw the melting curve (see Figure 4). The melting curve peaks of the standard and samples were single, and the melting temperature of the standard was the same (74.5±0.5°C), indicating that the amplification reaction product had a uniform melting temperature, good specificity, and no primer-dimer.

According to the change of fluorescence signal and the standard curve, the gene abundance of PD-L2 in the sample DNA can be measured.

Example 3 : Sensitivity, specificity and repeatability analysis of porcine PD-L2 real-time fluorescent quantitative PCR system

The porcine PD-L2 recombinant plasmid constructed in Example 1 was used as the positive recombinant standard plasmid; the porcine PD-L2 real-time fluorescent quantitative PCR detection system established in Example 2 was used; the real-time fluorescent quantitative PCR instrument was produced by LifeTechnologies (USA) ABI7500 fluorescent quantitative PCR instrument was used.

A 10-fold serial dilution of 10 ¹ to 10 ⁹ copies/μL porcine PD-L2 positive recombinant standard plasmid was used for a sensitivity test. The results showed that the detection limit of PD-L2 was 10 copies/μL.

The specificity analysis of the real-time-PCR melting curve of porcine PD-L2 molecule (see Figure 4) and agarose gel electrophoresis of the product showed that the melting curve had a single narrow peak, which could be amplified to the same extent as the expected target fragment. The size of the specific fragment, no non-specific products and primer dimers (see Figure 5).

The intra-assay and inter-assay repeatability tests were carried out with the established method, and the results showed that the intra-assay and inter-assay coefficients of variation were less than 3%, and the repeatability was good (see Table 1).

Table 1 Intra-assay and inter-assay reproducibility evaluation results of porcine PD-L2 real-time fluorescent quantitative PCR method

Example 4 : Using the real-time fluorescence quantitative PCR method established by the present invention, the variation law of the PD-L2 gene abundance in peripheral blood mononuclear lymphocytes of pigs after viral disease infection was analyzed.

The porcine PD-L2 real-time fluorescent quantitative PCR system established in Example 2 was used; the real-time fluorescent quantitative PCR instrument was ABI7500 fluorescent quantitative PCR instrument produced by Life Technologies (USA).

Fifteen 30-day-old healthy piglets were raised to 45-day-old, tested with IDEXX swine fever virus antibody detection kit, and confirmed that the CSFV antibody was negative, and were randomly divided into a control group (5 piglets) and an experimental group (10 piglets). The experimental group and the control group were kept in isolation, and the experimental group was injected with 0.1 mL/head of CSFV dilution through the neck muscle; the control group was treated with sterilized PBS for the same treatment.

Peripheral venous blood was collected on days 0, 3, 7, 10, 14, and 2 after CSFV infection of 45-day-old piglets, and piglets of the same age that were not infected with CSFV were used as controls. Nuclear lymphocytes, and total RNA was extracted, and cDNA was synthesized by reverse transcription using a reverse transcription kit (Dalian Bao Biology), using the specific primers provided in Example 1, and using the real-time fluorescent quantitative PCR system established in Example 2 for detection. The PD-L2 gene copy number (ie, gene abundance) in the peripheral blood mononuclear lymphocytes of the infection group and the control group can be obtained by calculating according to the standard curve, as shown in Figure 6.

The results showed that compared with healthy piglets not infected with CSFV, the abundance of PD-L2 gene was different at different stages after CSFV infection due to the different immune status of the body. Compared with the control group (P<0.05), it increased significantly (P<0.05) on the 3rd day; continued to increase on the 7th day, which was extremely significant compared with the control group (P<0.01), and began to decrease on the 10th day, but Compared with the control group, it was still significant (P<0.05), and then continued to decrease without statistical significance. It can be seen that the abundance of porcine PD-L2 gene has a greater correlation with the course of classical swine fever.

Example 5: The relationship between the change of PD-L2 expression and the viral load in plasma after swine fever virus (CSFV) infection

Primers and probes for real-time fluorescent quantitative PCR of CSFV load were synthesized by a biological company. The sequences of primers and probes are as follows:

Forward primer F: 5'-CCCTGAAGTGGATTAGAA-3',

Reverse primer R: 5'-TACCCTTGTTGATCCTATC-3',

TaqManprobe: 5'-(FAM)TTCACCGACTGTCCATTGTGG(Eclipse)-3'

The peripheral blood was collected on the 0, 3, 7, 10, 14 and 21 days after CSFV infection of 45-day-old piglets, and the total RNA of CSFV in the peripheral blood was extracted, and the reverse transcription kit (Dalian Baobio) was used to reverse transcribe Synthesize cDNA. The reverse transcription reaction system was: 2 μL 5×PrimeScriptBuffer, 0.5 μL PrimeScriptRTEnzymeMixI, 0.5 μL LigodT primer, 0.5 μL Random6mers, 1 μg total RNA and RNaseFreedH2O. The reverse transcription reaction conditions were: 37°C for 15 minutes, 85°C for 5 seconds, and the synthesized cDNA was stored at -20°C for future use. Real-time fluorescent quantitative PCR was used to detect the load of CSFV, the reagent was premixExTaq (probeqPCR) (Dalian Bao Biology), and it was carried out on ABI7500 instrument. The real-time fluorescent quantitative PCR reaction system was 20 μL system: 10 μL PremixExTaq, 0.4 μL CSFV forward primer (10 μmol/L), 0.4 μL reverse primer (10 μmol/L), 0.8 μL TaqMan probe (10 μmol/L), 0.4 μL ROXPreferenceDye, 2 μL template DNA and 6 μL triple distilled water. The reaction system was: hot start at 95°C for 10 seconds, 45 cycles, denaturation at 95°C for 15 seconds, annealing and extension at 56°C for 45 seconds. The load of CSFV in plasma was calculated according to the standard curve.

The results showed that the viral load began to increase on the 3rd day after CSFV infection, reached a peak on the 7th day, and then gradually decreased (see Figure 7).

Combining the results of Example 4, it can be seen that with the replication of the virus in the body, the expression level of PD-L2 gradually increases, indicating that the infection of CSFV and the viral load are closely related to the changes in the abundance of the porcine PD-L2 gene.

Example 6: Relationship between PD-L2 expression changes and IL-2, IL-10 cytokine expression changes after porcine CSFV infection

Using the porcine PD-L2 real-time fluorescent quantitative PCR system established in Example 2, adopting the peripheral blood of 0, 3, 7, 10, 14 and 21 days after the CSFV infection of Example 4 of 45-day-old piglets, and using its peripheral blood list The total RNA of nuclear lymphocytes was reverse-transcribed into cDNA as a template, and the expression changes of porcine cytokines IL-2 and IL-10 were detected on days 0, 3, 7, 10, 14 and 21 after CSFV infection.

The real-time fluorescent quantitative PCR instrument was carried out by ABI7500 fluorescent quantitative PCR instrument produced by Life Technologies (USA).

The results showed that the expression level of IL-2 mRNA decreased on the 7th day of CSFV infection (P<0.05), and when the expression of PD-L2 increased (7th day), the expression level of IL-10 increased and the change was extremely significant (P<0.05). 0.01), see Figure 8.

After CSFV infection, the expression of PD-L2 increased, activating the PD-L2:PD-Ls signaling pathway, resulting in a decrease in IL-2 expression (day 7), see Figure 9. At the same time, the expression of IL-10 increased (day 7), indicating that the expression of IL-10 affected the expression of PD-L2 after CSFV infection. IL-10 is an immunosuppressive cytokine, so after CSFV infection, the expression of PD-L2 in pigs increased, which activated the PD-L2:PD-Ls signaling pathway, and the reduction of IL-2 and the expression of IL-10 Elevated, so that the body is in a state of immunosuppression.

SEQUENCELISTING

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Claims (2)

1. A method for constructing a positive standard recombinant plasmid for PD-L2 real-time fluorescent quantitative PCR of porcine peripheral blood mononuclear lymphocytes, characterized in that: the method comprises the following steps: Collect the peripheral blood of piglets, isolate the peripheral blood mononuclear lymphocytes, extract the total RNA of the peripheral blood mononuclear lymphocytes, reverse transcribe the total RNA into cDNA, store the cDNA at -20°C, and amplify PD by common PCR method - L2 target gene fragment; Amplify the PD-L2 target gene fragment by common PCR method, detect the target gene fragment by agarose gel electrophoresis, recover and purify; then connect the purified PD-L2 target gene fragment with the pMD18-T vector, and transform it into a competent state In DH5α cells, the recombinant plasmid was extracted, cloned and screened for sequencing analysis, and a positive standard recombinant plasmid with the same sequence as the target gene fragment was obtained; The reaction system for ordinary PCR amplification is 25 μL, and the reaction system is as follows: 2 μL sample cDNA template, 2.5 μL 10×PCRBuffer, 2 μL dNTP, 0.5 μL each of the forward primer and reverse primer at a concentration of 20 μmol/L, and 0.5 μL of TaqDNA Polymerase, the rest is sterile triple-distilled water; general PCR reaction procedure: hot start at 95°C for 1 min; denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s, 30 cycles, and finally extension at 72°C for 10 min; Wherein, the forward primer F is: 5'-GCCAACACCAGCTATACCAAGAC-3', The reverse primer R is: 5'-GGGATGAAAACACGGAGCA-3'. 2. The construction method according to claim 1, characterized in that: the target gene fragment is the sequence represented by SEQ ID No.1.