CN109207577B - Application of MARCO in screening of porcine reproductive and respiratory syndrome resistant pigs - Google Patents
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Abstract
The invention discloses an application of MARCO in screening of anti-blue ear disease pigs. The research of the invention shows that the expression quantity of MARCO in a pig body is closely related to PRRSV infection, the MARCO expression is inhibited after PRRSV is infected with PAMs, the MARCO interference promotes the PRRSV replication, and the MARCO overexpression inhibits the virus replication; when the expression level of MARCO is abnormally high in vivo, the swine has resistance to PRRSV, and vice versa. Therefore, MARCO can be used as a novel standard for judging PRRSV susceptibility or resistance, lays a solid foundation for identifying PRRSV susceptibility and PRRSV resistant pig screening and breeding, has good clinical application value for PRRSV resistant pig screening and breeding, has important significance for preventing and controlling porcine reproductive and respiratory syndrome, and is also beneficial to breeding of excellent strain pigs.
Description
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, relates to the application of MARCO in screening anti-blue ear disease pigs.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as Porcine reproductive and respiratory syndrome virus, is caused by PRRSV. As early as 1992, the world animal health organization (Office International Des epidemics, OIE) classified the disease as a class B infectious disease. Currently, PRRS is one of the most important infectious diseases in the swine industry, is distributed in almost all swine countries, and causes huge economic losses to the swine industry all over the world. In 2006, the Pig High Fever (PHFS) developed in most provinces and vietnam of China caused a huge economic loss to the pig industry, and the disease was caused by a variant PRRSV, named a Highly pathogenic Porcine reproductive and respiratory syndrome virus (HP-PRRSV). HP-PRRSV is characterized by high fever, high pathogenicity, and high mortality. Currently, the department of agriculture has placed this disease in a forced immunization program for major animal epidemic.
The prevention and control of PRRSV is a difficult problem in China and even the world at present. The difficulty in controlling the PRRSV is mainly shown in the following aspects: (1) macrophage-tropic and immunosuppressive diseases, PRRSV primarily infects Porcine Alveolar Macrophages (PAMs), PAMs are immune cells that destroy PAMs, thereby destroying the body's immune system, causing immunosuppression; (2) antigen variability, PRRSV mutation is relatively fast at present, the use of attenuated vaccine is one reason for promoting virus mutation, recently there are literature reports, new strain NADC30 of blue ear appears in the United states, China also separates new strain similar to the United states, named NADC30-like, and another literature report separates PRRSV pathogenic strain highly homologous with vaccine virus genome from pig farm, and the virulence is enhanced, the analysis may be vaccine virus resistance or recombination and virus dispersion; (3) the vaccine has no cross protection, the PRRSV vaccine in the current market almost has no cross protection, and different strains have no cross protection; (4) the dependence of the antibody is enhanced, the infection of PRRSV can stimulate the organism to generate the antibody, but the high-titer antibody can not neutralize the virus but promote the proliferation of the virus; (5) the virus is continuously infected, after PRRSV is infected, viremia can be detected in a pig body for a long time, and the PRRSV lasts for 5 months in the pig body; (6) the mixed infection is common to the blue ear and other diseases clinically at present, and particularly the mixed infection of the circovirus, haemophilus parasuis, swine plague and the like and the PRRSV, so that the prevention and control of the PRRSV are difficult.
In addition, because the genetic background is closely related to biological disease resistance, the work of screening the porcine reproductive and respiratory syndrome resistant pigs based on a gene means has incomparable advantages for the prevention and control of the disease, and the important point is to find out genes related to resistance or susceptibility, so that not only can the infection mechanism of the porcine reproductive and respiratory syndrome be researched, but also a new method is provided for the treatment and prevention of the porcine reproductive and respiratory syndrome.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing control technology of the porcine reproductive and respiratory syndrome and provides a gene related to resistance of the porcine reproductive and respiratory syndrome. The expression quantity of MARCO is proved to be closely related to the reproduction of PRRSV, so that the method is expected to become a novel method for screening the PRRSV resistant pigs and has good clinical application value for the cultivation of the PRRSV resistant pigs.
The invention aims to provide application of MARCO in screening of anti-blue ear disease pigs.
It is another object of the present invention to provide a method for determining resistance or susceptibility to blue ear disease in swine.
It is a further object of the present invention to provide the use of MARCO for increasing resistance of swine to porcine reproductive and respiratory syndrome virus, or for the preparation of a medicament or formulation for increasing resistance of swine to porcine reproductive and respiratory syndrome virus.
It is a further object of the present invention to provide the use of MARCO for breeding porcine breeds resistant to porcine reproductive and respiratory syndrome viruses.
The above purpose of the invention is realized by the following technical scheme:
the research of the invention finds that the MARCO expression is inhibited by PRRSV infected PAMs cells in pigs at different time points and different infection numbers. That is, the expression quantity of MARCO is closely related to PRRSV infection, and has close relation with the susceptibility of pigs to the porcine reproductive and respiratory syndrome, and the PRRSV replication can be promoted after the expression of MARCO is interfered by siRNA, and the PRRSV replication can be inhibited after MARCO overexpression. Namely, the MARCO expression level in the pig body is higher, so that the pig body has resistance to the porcine reproductive and respiratory syndrome virus; otherwise the susceptibility is higher.
Therefore, the following applications should be within the scope of the present invention:
application of MARCO in screening anti-blue ear disease pigs.
Gene ID of the pig MARCO: 100516298.
the porcine reproductive and respiratory syndrome virus comprises a classical strain and a highly pathogenic strain (HP-PRRSV).
Use of MARCO for increasing resistance of pigs to porcine reproductive and respiratory syndrome virus.
The application of MARCO in preparing medicines or preparations for improving the resistance of pigs to the porcine reproductive and respiratory syndrome virus.
Application of MARCO in screening and/or breeding porcine breeds with resistance to the porcine reproductive and respiratory syndrome virus.
Use of a MARCO expression activator (e.g. overexpression) for increasing resistance in pigs to porcine reproductive and respiratory syndrome virus.
The MARCO expression activator is applied to the preparation of the drug or the preparation for improving the resistance of the pig to the porcine reproductive and respiratory syndrome virus.
In addition, the invention also provides a method for identifying the susceptibility of the pig to the porcine reproductive and respiratory syndrome, and particularly relates to identifying the resistance or the susceptibility of the pig to the porcine reproductive and respiratory syndrome by detecting the expression level (expression quantity) of MARCO in the pig body.
The criteria for the identification are: when the expression amount of MARCO is high, the pig has resistance to the porcine reproductive and respiratory syndrome virus; otherwise, the disease is susceptible.
In the specific operation, the expression level of MARCO is based on the general level of MARCO expression level in pigs in the field of technology. The levels described herein are relative and are based on higher or lower MARCO expression abnormalities to determine whether a pig is likely to be resistant to PRRSV, or susceptible to PRRSV.
More specifically, based on the results of the experiments of the present invention, the specific data criteria identified were roughly: when the expression level of MARCO is higher than 2.5 +/-0.5X 10-2(relative to the internal reference HPRT 1), swine are resistant to the virus; the expression level is less than 2.5 +/-0.5X 10-2(relative to internal reference HPRT 1).
The method for detecting the expression level of MARCO in the present invention is not limited strictly, and may be a method for detecting the expression level of a gene existing in the art.
As a preferable embodiment, the invention provides a primer group for detecting the expression level of pig MARCO, which comprises an upstream primer TREM2-F and a downstream primer TREM2-R, wherein the sequences are respectively shown in SEQ ID NO. 1-2.
The application of the primer group in judging the resistance or susceptibility of the pig to the blue-ear disease or in screening the blue-ear disease resistant pig is also within the protection scope of the invention.
The invention selects the application of different pig breeds in researching MARCO in screening the blue ear disease resistant pigs. Selecting local pig of China, such as Tongcheng pig, Meishan pig (not susceptible to blue-ear disease) and Changbai pig, and Dabai pig (susceptible to blue-ear disease), respectively, performing PRRSV challenge, dissecting, and collecting virus titer TCID in serum, lung and lymph node tissue50And determining the PRRSV infectivity difference of the Tongcheng pigs, Meishan pigs, foreign breed Changbai pigs and big white pigs according to the PRRSV N protein expression level, and researching the PRRSV infectivity relation with the 4 breed pigs according to the MARCO expression level. The result shows that the method for screening the anti-blue ear disease pig by using the MARCO is reliable and accurate.
The invention has the following beneficial effects:
according to the invention, the expression quantity of MARCO in a pig body is closely related to PRRSV infection through first research, and the MARCO can be used as a novel method for judging PRRSV resistance or susceptibility; when the expression level of MARCO is abnormally high, the pigs have resistance to PRRSV, and are susceptible to the contrary.
The discovery of the invention lays a foundation for screening the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) resistant pigs and breeding the PRRSV resistant pigs, has good clinical application value for screening and breeding the PRRSV resistant pigs, has important significance for preventing and controlling the porcine reproductive and respiratory syndrome, and is also beneficial to breeding of excellent strain pigs. Meanwhile, the invention also provides a new application for MARCO.
Drawings
FIG. 1 shows the difference in MARCO expression in different tissues (lung, spleen, liver, kidney).
FIG. 2 shows MARCO mRNA expression levels at different time points in PRRSV infected PAMs cells.
FIG. 3 shows the expression level of MARCO protein at different time points when PRRSV infects PAMs cells.
FIG. 4 shows the expression levels of MARCO mRNA in PRRSV infected PAMs cells at different multiplicity of infection.
FIG. 5 shows MARCO mRNA expression levels after siRNA interference in MARCO.
FIG. 6 shows the expression level of PRRSV N protein mRNA after siRNA interference of MARCO.
FIG. 7 shows the level of PRRSV N protein expression after MARCO overexpression. MYC is the MARCO tag.
FIG. 8 shows the level of PRRSV N protein mRNA expression after MARCO overexpression.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Statistical analysis of the following examples of the invention: all experiments were repeated at least 3 times independently, with results expressed as mean and standard error, using one-way analysis of variance and T-test analysis. All statistical analyses used a P <0.05 as a test standard with significant statistical differences, SPSS 16.0 and GraphPad Prism 5 as analytical software.
EXAMPLE 1 PAMs cell isolation and culture
1. After the weaned piglets which are 6-8 weeks old and free of specific pathogen infection such as PRRSV, porcine circovirus type 2 (PCV-2), Classical Swine Fever Virus (CSFV) and the like are bound, the anterior vena cava is exsanguinated to die, the trachea is ligated, the chest is cut open, the complete lung is taken out together with the heart, the surface of the lung is fully rinsed by sterile PBS liquid, blood clots and dirt are removed, and the heart is removed after the completion of the removal. Pouring into PBS buffer solution, gently tapping and kneading lung surface for 5-10 min, and recovering bronchoalveolar lavage fluid. Repeat steps 2 to 3 times until a total of about 1000 mL of lavage fluid is recovered. The recovered alveolar lavage fluid was gently pipetted back and forth to separate cell aggregates, centrifuged at 1000 rpm for 10 min at 4 ℃ and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium, centrifuged at 1000 rpm for 5 min at 4 ℃ and the supernatant discarded. With 20% FBSGently blowing and beating the resuspended cells with RPMI-1640 nutrient solution, adding double antibodies to prevent operation pollution, uniformly separating the separated cells into culture plates, and culturing at 37 deg.C and 5% CO2Culturing in a humidifying incubator.
2. And observing the morphology and activity of the PAMs cells, and storing the PAMs cells in liquid nitrogen for later use.
Example 2 study of MARCO Gene to modulate PRRSV replication in vitro
1. Culturing PAMs cells by RPMI 1640 culture solution containing 10% fetal calf serum, inoculating the virus PRRSV with MOI =0.1, continuously culturing in RPMI 1640 culture solution containing 2% fetal calf serum at 37 ℃, collecting cells at different time points, and respectively carrying out qRT-PCR and Western-blot detection on MARCO genes. The results are shown in fig. 2 and fig. 3, and the expression level of MARCO is obviously reduced after the PRRSV infects the PAMs cells.
2. Culturing PAMs cells by RPMI 1640 culture solution containing 10% fetal calf serum, inoculating PRRSV by MOI with different infection numbers, continuously culturing for 24 h at 37 ℃ in RPMI 1640 culture solution containing 2% fetal calf serum, collecting cells, and carrying out qRT-PCR detection on MARCO genes. The results are shown in fig. 4, and the expression level of MARCO is remarkably reduced after PRRSV infects PAMs cells.
Example 3 MARCO interference
1. The designed MARCO siRNA and 1 pair of Negative controls were synthesized by Thermo Fisher Scientific using Lipofectamine ™ RNAIAMAX transfection reagent (available from Thermo Fisher Scientific). PAMs cells were cultured in 2 6-well plates with 10% fetal bovine serum in RPMI 1640 medium to 60-70% density, and siRNA (3 pairs of siRNA and 1 control) and Lipofectamine ™ RNAiMAX were applied using Opti-MEM®Diluting Reduced Serum Medium, mixing at a ratio of 1:1, culturing at room temperature for 5 min, adding into PAMs cells replaced with fresh RPMI 1640 culture Medium at 37 deg.C and 5% CO2Culturing for 6 h in an incubator, discarding the supernatant, washing with PBS for 1 time, continuously culturing for 48 h with RPMI 1640 culture solution containing 10% fetal calf serum, collecting cells, and verifying the MARCO interference effect through qRT-PCR experiment. After another 6-well plate is cultured for 48 h, PRRSV is added into the culture medium, the culture medium is cultured for 24 h, and cells are collected to verify the influence of MARCO interference on PRRSV replication through a qRT-PCR experiment.
2. The results are shown in fig. 5, where siRNA significantly inhibited MARCO mRNA expression relative to the control group after MARCO was interfered with siRNA.
After MARCO interference, PRRSV was detoxified, and the results are shown in fig. 6, where PRRSV N protein mRNA levels were significantly increased after siRNA treatment relative to the control group.
Taken together, MARCO promoted PRRSV replication after siRNA interference.
Example 4 MARCO overexpression
1. Construction of pcDNA3.1-MARCO overexpression vector, culturing PAMs cells to 60-70% density in 2 6-well plates in RPMI 1640 medium containing 10% fetal bovine serum, transferring pcDNA3.1-MARCO and pcDNA3.1 empty vectors into Marc-145 cells using Lipofectamine [. sup.2000 (available from Thermo Fisher Scientific Co., Ltd.), respectively, at 37 ℃ and 5% CO2Culturing for 6 h in an incubator, discarding the supernatant, washing with PBS for 1 time, culturing for 48 h with RPMI 1640 culture solution containing 10% fetal calf serum, collecting cells, and detecting the over-expression effect. After another 6-well plate is cultured for 48 h, PRRSV is added into the culture medium, and then cultured for 24 h, and cells are collected to detect the influence of over-expression of MARCO on PRRSV replication.
2. The results are shown in FIG. 7, MYC is expressed after MARCO overexpression, which indicates that MARCO overexpression is successful; meanwhile, the PRRSV N protein expression is obviously inhibited.
After MARCO overexpression, PRRSV was detoxified, and the results are shown in fig. 8, with significantly reduced PRRSV N protein mRNA levels relative to the control.
Taken together, MARCO over-expression inhibits PRRSV replication.
Example 5 application of MARCO in screening of anti-blue ear disease pigs
1. The method for screening the blue ear disease resistant pigs by using the MARCO is verified by selecting local domestic pig breeds of Tongcheng pigs, Meishan pigs and foreign breeds of Changbai pigs and big white pigs as experimental objects.
Among them, it is known that the pigs in Tongcheng and Meishan are not susceptible to, i.e., resistant to, the porcine reproductive and respiratory syndrome virus; the Changbai pigs and the big white pigs are more susceptible to the porcine reproductive and respiratory syndrome virus.
2. Experimental methods
Selecting 6 pigs of Tongcheng pigs, Meishan pigs, Changbai pigs and Dabai pigs respectively, dividing 1 group of 3 pigs into 2 groups, wherein 1 group is subjected to PRRSV virus challenge, and the other 1 group is not subjected to any treatment (contrast), collecting blood respectively at the 7 th, 14 th, 21 th and 28 th days before and after virus challenge, synchronously collecting blood in the contrast group, killing the 28 th day, and collecting lung and lymph node tissues.
Taking a part of serum for TCID50And (3) measuring, extracting RNA from the residual serum and lung and lymph node tissues, reversing, carrying out qRT-PCR detection on MARCO and PRRSV N proteins, and carrying out pathological section on the lung tissues.
According to TCID50And determining the PRRSV infectivity of the Tongcheng pigs, Meishan pigs, and foreign breeds of Changbai pigs and big white pigs by the PRRSV N protein expression level and lung tissue pathological section, and researching the relation between the PRRSV infectivity and 4 breeds of pigs according to the MARCO expression level.
3. According to the results, it can be judged that the Tongcheng pigs and Meishan pigs are less susceptible to the porcine reproductive and respiratory syndrome, while the Changbai pigs and Dabai pigs are more susceptible to the porcine reproductive and respiratory syndrome.
Meanwhile, the detection result of the MARCO expression level shows that the MARCO expression level in the Tongcheng pigs and the Meishan pigs is obviously higher than that of the Changbai pigs and the big white pigs.
In addition, based on a large number of experimental results, the specific data standard for identifying the susceptibility of pigs to the porcine reproductive and respiratory syndrome virus is approximately as follows: when the expression level of MARCO is higher than 2.5 +/-0.5X 10-2(relative to the internal reference HPRT 1), swine are resistant to the virus; the expression level is less than 2.5 +/-0.5X 10-2(relative to internal reference HPRT 1).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Zhongshan university
Application of <120> MARCO in screening of anti-blue ear disease pigs
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> upstream primer MARCO-F (forward primer MARCO-F)
<400> 1
gcagcgggta gacaacttc 19
<210> 2
<211> 20
<212> DNA
<213> downstream primer MARCO-R (reverse primer MARCO-R)
<400> 2
tgttgctcca tcttgtcccg 20
Claims (5)
1. The application of the reagent for detecting the expression level of the MARCO gene in preparing the reagent for screening the porcine reproductive and respiratory syndrome resistant pig is characterized in that the screening standard is that the higher the expression level of the MARCO gene is, the stronger the resistance of the pig to the porcine reproductive and respiratory syndrome virus is; the stronger the susceptibility, conversely.
2. The application of a reagent for detecting the expression level of the MARCO gene in preparing products for detecting and judging susceptibility or resistance of pigs to the porcine reproductive and respiratory syndrome virus is characterized in that the detection and judgment standard is that the higher the expression level of the MARCO gene is, the stronger the resistance of the pigs to the porcine reproductive and respiratory syndrome virus is; the stronger the susceptibility, conversely.
3. The use of claim 1 or 2, wherein the reagent for detecting the expression level of the MARCO gene is a primer set represented by SEQ ID No. 1-2.
4. A method for screening anti-blue ear disease pigs is characterized in that the resistance or susceptibility of the pigs to blue ear virus is identified by detecting the expression level of MARCO in the pigs; the screening standard is that the resistance of pigs with higher MARCO expression quantity to the porcine reproductive and respiratory syndrome virus is stronger; the stronger the susceptibility, conversely.
5. The method of claim 4, wherein the detection of the expression level of MARCO in the pig is performed by qRT-PCR, wherein the primer set comprises an upstream primer TREM2-F and a downstream primer TREM2-R, and the sequences are respectively shown in SEQ ID NO. 1-2.
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| PCT/CN2018/099287 WO2020024313A1 (en) | 2018-07-31 | 2018-08-08 | Application of marco in screening prrsv-resistant pigs |
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| CN117604167A (en) * | 2023-11-23 | 2024-02-27 | 扬州大学 | PMA-qPCR universal detection kit and triple identification kit for infectious PRRSV |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878104A (en) * | 2015-06-01 | 2015-09-02 | 北京泱深生物信息技术有限公司 | Bile duct cancer diagnosis and treatment molecular marker and application thereof |
| CN106244682A (en) * | 2016-07-29 | 2016-12-21 | 中山大学 | TREM 2 gene is differentiating that pig is to the application in reproductive and respiratory syndrome poison susceptibility |
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| CN104878104A (en) * | 2015-06-01 | 2015-09-02 | 北京泱深生物信息技术有限公司 | Bile duct cancer diagnosis and treatment molecular marker and application thereof |
| CN106244682A (en) * | 2016-07-29 | 2016-12-21 | 中山大学 | TREM 2 gene is differentiating that pig is to the application in reproductive and respiratory syndrome poison susceptibility |
Non-Patent Citations (1)
| Title |
|---|
| 猪A类清道夫受体SRA和MARCO的克隆、表达及其介导对病原菌吞噬功能的研究;黎倩倩;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160415(第4期);第11页、表1-1 * |
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