CN114470160A

CN114470160A - Inhibitors of viral replication and uses thereof

Info

Publication number: CN114470160A
Application number: CN202210268217.5A
Authority: CN
Inventors: 成子强; 杨剑豪; 周德方; 张利; 周静
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-05-13
Anticipated expiration: 2042-03-18
Also published as: CN114470160B

Abstract

The invention discloses a virus replication inhibitor and application thereof, belonging to the technical field of molecular immunology and virology. The invention discovers for the first time that serum amyloid A can be used as a virus replication inhibitor, can directly and obviously inhibit MDV replication, reduces the virus load by more than 3 times compared with a control group when collecting viruses, and has no toxic or side effect on cells. The serum amyloid A can be widely used as an anti-MD drug in the production practice of poultry industry defense MDV, and has great economic value and research significance.

Description

Inhibitors of viral replication and uses thereof

Technical Field

The invention relates to the technical field of molecular immunology and virology, in particular to a virus replication inhibitor and application thereof.

Background

Marek's Disease (MD) is a malignant lymphoid tumor Disease of chickens caused by Marek's Disease Virus (MDV) and can also cause immunosuppression of infected chickens. The Jozef Mark, Hungarian pathologist in 1907, first discovered that sciatic nerve enlargement and paralysis of the rooster resulted from infiltration of monocytes under the peripheral nerve and spinal nerve roots. In 1961, the name Marek's disease began to be used under the initiative of Biggs, a scientist in England. Marek's disease virus was isolated by scholars in the United kingdom in 1967. Since the last 70 s, various MDV vaccines have been widely used in poultry farming. The disease is still a major threat to the healthy development of the poultry industry worldwide. In 1984, sick chickens in the united states alone lost $ 1200 million due to MDV direct death and abandonment by farmers, with the cost of vaccines and their vaccination and reduction in egg production, lost $ 1.69 million and worldwide lost $ 9.43 million due to marek's disease. With the wide use of various MDV vaccine preparations, the recombination of wild strains and vaccine strains occurs in Marek's virus, the toxicity of the vaccine strains is enhanced, MDV vaccine failure frequently occurs, and the problem that the production performance of chicken groups infected with polluted vaccine strains and novel wild strains is seriously reduced is increasingly highlighted. Vaccines cannot protect against the new MDV, and the like, and the control of Marek's disease presents a serious challenge. There is an urgent need in production for biologicals that prevent MDV infection and reduce the impact of MDV on the performance of chicken flocks.

The innate immune response is the primary barrier of the host against invasion of pathogenic microorganisms, and when pathogenic microorganisms invade the organism, the contained physical barrier, acute reaction phase protein and specific immune response sequentially play roles in resisting invasion of pathogenic microorganisms. When pathogenic microorganisms invade, a plurality of acute reaction period proteins can be rapidly and greatly increased and play various direct or indirect antiviral roles, for example, the proteins are directly combined with viruses to inhibit the attachment or the replication of the viruses, enhance the immunity of a host body, create a biological environment which is not beneficial to the survival of the pathogenic microorganisms, and the like to enhance the disease resistance of the body. Meanwhile, the innate immunity protein is generated by the host, the host can not generate strong rejection reaction to the innate immunity protein, and the innate immunity protein has huge development and application prospects in the field of antivirus.

The research and development of the innate immunity protein for preventing and inhibiting MDV infection and reducing the influence of MDV on the production performance have important and urgent practical significance for the prevention and control of MD.

Disclosure of Invention

In view of the above prior art, the present invention aims to provide a virus replication inhibitor and its application. The invention discovers that Serum Amyloid A (SAA) can be used as a virus replication inhibitor, can directly and obviously inhibit MDV replication, and has no toxic or side effect on cells; can be developed into MDV treatment medicine and has wide application prospect.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a use of serum amyloid a in the following (1) or (2):

(1) preparing a medicament for preventing and/or treating diseases caused by Marek's disease virus;

(2) preparing a medicament for inhibiting the replication of Marek's disease virus;

the serum amyloid A is a protein represented by any one of the following proteins:

A1) protein with an amino acid sequence of SEQ ID NO. 1;

A2) the fusion protein is obtained by connecting labels at the N end or/and the C end of the amino acid sequence shown in SEQ ID NO. 1.

Wherein, the proteins A1) and A2) can be artificially synthesized, or can be obtained by synthesizing the coding genes and then carrying out biological expression.

In the above proteins, the protein tag refers to a polypeptide or protein that is expressed by fusion with a target protein by using a DNA in vitro recombination technology, so as to facilitate expression, detection, tracing and/or purification of the target protein. Wherein a tag may be attached to the amino terminus or the carboxyl terminus of the protein of (A1) in order to facilitate purification of the protein of (A1). The tag may be Poly-Arg (typically 6 RRRRR), Poly-His (typically 6 HHHHHHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).

In a second aspect of the present invention, there is provided a use of a gene encoding serum amyloid A in (1) or (2) below:

(2) preparing the drug for inhibiting the replication of Marek's disease virus.

In the above application, the gene encoding serum amyloid A is a nucleic acid molecule represented by i) or ii) or iii) as follows:

i) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 2;

ii) a nucleic acid molecule which is 90% or more than 90% identical to the nucleotide sequence of i) and expresses a protein having the same function;

iii) a nucleic acid molecule other than i) which codes for the amino acid sequence shown in SEQ ID NO. 1.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed using computer software, for example, as determined using the BLAST algorithm (Altschul et al 1990.journal of Molecular Biology 215: 403-.

In the above nucleic acid molecule, the 90% or more identity may be at least 90%, 91%, 92%, 95%, 96%, 98% or 99% identity.

In a third aspect of the present invention, there is provided a use of a biomaterial related to serum amyloid a in (1) or (2) below:

(2) preparing the drug for inhibiting the replication of Marek's disease virus.

In the above application, the biological material related to serum amyloid a is any one of the following:

B1) a recombinant expression vector containing a serum amyloid A coding gene;

B2) a recombinant microorganism comprising a gene encoding serum amyloid A;

B3) a transgenic cell line comprising a gene encoding serum amyloid A;

the nucleotide sequence of the serum amyloid A coding gene is shown as SEQ ID NO. 2.

In the above applications, a recombinant expression vector containing a serum amyloid A-encoding gene can be constructed using an existing expression vector. The existing expression vector may be a eukaryotic expression vector, such as a PEX-3 vector (pGCMV/MCS/Neo); prokaryotic expression vectors, such as pET-22b (+) vector plasmids, are also possible. When a recombinant expression vector is constructed using the serum amyloid a encoding gene of the present invention, enhancers including a translation enhancer, a transcription enhancer, and the like may also be used.

In the above applications, the recombinant microorganism may be a virus (e.g., lentivirus), yeast, bacterium, algae, fungus, or the like.

In a fourth aspect of the present invention, there is provided a medicament for inhibiting replication of Marek's disease virus, said medicament comprising an effective amount of serum amyloid A as an active ingredient.

Preferably, the concentration of serum amyloid A in the medicament is 5 μ g/mL-10 μ g/mL.

Further, the medicine can also contain: one or more of a substance for enhancing or increasing the activity of serum amyloid A, a substance for increasing the content of serum amyloid A and a substance for increasing the expression level of a gene encoding serum amyloid A.

The invention has the beneficial effects that:

the invention discovers for the first time that the serum amyloid A can be used as a virus replication inhibitor, can directly and obviously inhibit MDV replication, reduces the virus load by more than 3 times compared with a control group when collecting viruses, and has no toxic or side effect on cells.

The serum amyloid A can be widely used as an anti-MD drug in the production practice of poultry industry defense MDV, and has great economic value and research significance.

Drawings

FIG. 1: schematic representation relating to expression of MDV-activated SAA;

a: MDV virus titer plots at various time periods after infection with MDV; b: a graph of MDV meq protein expression at various time periods following infection with MDV; c: mRNA expression level profiles of SAA at different time periods after infection with MDV.

FIG. 2: the correlation plot of significant increase in MDV load for over-expression of SAA,

a: after transfecting the no-load plasmid and the SAA overexpression plasmid, infecting MDV to maintain the mRNA expression amount of the SAA after 36 h; B. c, D: after transfection of the empty plasmid and SAA overexpression plasmid, MDV was maintained 36h after infection with MDV for viral titer (C), MDV meq protein mRNA expression (B) and protein expression (D).

FIG. 3: interference with SAA significantly facilitates the replication of MDV;

e: after transfection of disordered interfering RNA and SAA interfering RNA, maintaining the mRNA expression level of SAA after 36h after infection of MDV; F. g and H: after transfection of the disordered interfering RNA and SAA interfering RNA, the virus titer (G) of MDV, the mRNA expression level (F) of MDV meq protein and the protein expression level (H) of MDV were maintained after 36H of MDV infection.

FIG. 4 is a schematic diagram of pET22b-his-SAA recombinant protein expression plasmid constructed according to the present invention.

FIG. 5 is a SDS-PAGE detection of the purified recombinant SAA of the present invention, and as shown in FIG. 5, the purification of the recombinant SAA was very good with only a single band.

FIG. 6 is a standard curve drawn by the BCA protein quantitative determination kit.

FIG. 7 is a graph showing the cytotoxicity of CCK-8 on recombinant SAA after renaturation of the present invention, as shown in the graph, different concentrations of SAA do not produce great cytotoxicity on CEF cells,

FIG. 8 is a graph of the real-time fluorescent quantitative PCR detection result of the effect of different concentrations of recombinant SAA on MDV transcription on CEF cells after renaturation in 24h of the invention, as shown in FIG. 8, at 24h, the recombinant SAA can inhibit MDV transcription and has concentration dependence.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As mentioned previously, MD is one of the most important diseases facing the chicken industry. The main method adopted for preventing and treating MD at present is vaccine immunization. However, with the continuous increase of the toxicity of MDV, the MDV vaccine loses efficacy, and the problem that the production performance of chicken flocks infected with polluted vaccine strains and novel wild strains is seriously reduced becomes increasingly prominent.

Based on the above, in order to solve the problems in preventing and treating MD in the current chicken industry, the inventor develops an inhibitor capable of inhibiting MDV replication from innate immunity protein, and provides a new medicine source and a new prevention and treatment means for preventing and treating MD.

The inhibitor for inhibiting MDV replication of the present invention comprises Serum Amyloid A (SAA) as an active ingredient. The relative molecular weight of SAA protein is about 13KDa, 127 amino acids form exocrine type glycoprotein, the amino acid sequence is shown as SEQ ID NO.1, and the relative molecular weight is as follows:

MRLCICFVLL SIVLYASADN PFSSGIKFVR DAAGGARDMW RAYRDMREAN YIGADKYFHA RGNYDAARRG PGGAWAAKVI SDAREGWQSR VSGRGAEDTR LDQEANEWGR RGGDPNRFRP AGLPSKY。

the SAA protein has very low homology with the currently known proteins, and is relatively conserved among species. SAA is transcribed in the nucleus, activated and secreted into the cytosol, and finally into the extracellular matrix. In the prior art, reports related to MDV and serum amyloid A do not appear, and the research on the relation between the MDV and the serum amyloid A is in a blank state.

Based on the efforts of the inventor, the SAA has proved to have the function of resisting MDV replication through a large amount of experiments, and the preparation and the use method of the protein as an MDV replication inhibitor are established. Provides a theoretical and research basis for preventing subsequent production by using the polypeptide in practice, resisting MDV virus infection and reducing the influence of MDV on the production performance.

The specific idea of the invention is as follows:

the in vitro verification of the SAA expression level is carried out through a fluorescent quantitative PCR test, and the result shows that: MDV activates the expression of SAA; to verify whether SAA can inhibit MDV replication dependence, the inventors constructed SAA overexpression vectors. After CEF cells over-express SAA recombinant plasmids, MDV is inoculated, and the experimental result shows that: over-expression of SAA can reduce viral load in cells very significantly; in contrast, following SAA knockdown, the viral load of MDV increased significantly; we further pronucleus expressed SAA protein, cytotoxicity experiments prove that the pronucleus expressed SAA protein has no toxic effect on cells, and then the prokaryote expressed SAA protein is added into CEF cells to find that MDV replication is inhibited and has concentration dependence. The results show that: SAA has the effect of inhibiting MDV replication.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer.

The MDV used in the examples of the invention was a virulent strain Md5 of marek's disease virus, obtained from university of agriculture in shandong, which was obtained by the public from the applicant within 20 years from the filing date, for the purpose of repeating the invention.

Example 1: MDV-activated expression of SAA

To confirm the activation of SAA by MDV, this example establishes an in vitro cell model of MDV infection. Changes in SAA expression levels following MDV infection were detected by qPCR.

(1) MDV virus titer determination

Collecting cells at different time points after the MDV Md5 strain is inoculated, diluting the samples at different time points by 1000 times, inoculating the diluted samples into a six-well plate which is fully paved with a Chicken Embryo Fibroblast (CEF) single layer, repeating the dilution for 3 times, after inoculation for 96 hours, respectively counting the number of plaques in the wells with different dilution, and taking the average number of plaques, namely the virus titer at the time point.

The results are shown in FIG. 1 a; (a) MD5 infected CEF cells at different time points were extracted and, upon inoculation of normal CEF cells, the virus titer was calculated without time point MD 5.

FIG. 1a illustrates the reliability of the present embodiment for receiving virus as helper data.

(2)Western blot

Cells infected with Md5 for 6h, 12h, 24h, 36h, 48h, 72h were harvested, and radioimmunoprecipitation buffer (RIPA) and phenylmethylsulfonyl fluoride (PMSF) were mixed in 100: 1 by volume ratio to lyse cells; each protein sample was adjusted to a uniform concentration, and protein loading buffer was added and heated in 100 ℃ water for 5 min. Adding the sample into an electrophoresis apparatus, performing pressure stabilization on the concentrated gel at 80V, and performing pressure stabilization on the separated gel at 110V. After the electrophoresis is finished, transferring the protein on the protein gel to a PVDF membrane with the diameter of 0.22 mu m, sealing the membrane for 2h at 37 ℃ by using 5% skimmed milk powder, washing the membrane by using TBST, adding a mouse anti-MDV Meq protein antibody, incubating the membrane for 1h at 37 ℃, washing the membrane by using TBST, adding a sheep anti-mouse antibody, incubating the membrane for 1h at 37 ℃, and finally observing the membrane by using a Western blot developer.

As shown in FIG. 1b, (b) Western blot was used to detect the expression level of MDV Meq protein in MDV-infected cells and normal cells. The reliability of the present embodiment for receiving the poison can be illustrated by fig. 1 b.

(3) Fluorescent quantitative PCR/qPCR

Design qPCR specific primers: the sequence of the avian host protein SAA qPCR primer is as follows:

F:5’-TTCTCCAGCGGTATCAAGTT-3’，(SEQ ID NO.3)

R:5’-CCGAGCAGCATCGTAATT-3’；(SEQ ID NO.4)

the primer sequence of the avian reference gene GPADH is as follows:

F:5’-GAACATCATCCCAGCGTCCA-3’，(SEQ ID NO.5)

R:5’-CGGCAGGTCAGGTCAACAAC-3’。(SEQ ID NO.6)

the sequence of the Meq fluorescent quantitative primer is as follows:

F:5′-TTGTCATGAGCCAGTTTGCCCTAT-3′，(SEQ ID NO.7)

R:5′-TGAGGGAGGTGGAGGAGTGCAAAT-3′。(SEQ ID NO.8)

the primers were synthesized by Qingdao Hua Dagene Biotechnology GmbH.

The CEF cells infected with Md5 for 6h, 12h, 24h, 36h, 48h and 72h were harvested, total cellular RNA was extracted using a total cellular RNA extraction kit (QIAGEN, America), and then the RNA was reverse-transcribed into cDNA, and the expression change of SAAmRNA was detected by qPCR using this as a template.

The reaction system is as follows: 10 μ L SYBR Premix Ex TaqTM (2X), 0.4 μ L forward primer, 0.4 μ L reverse primer, 1 μ L cDNA, 8.2 μ L ddH in a 20 μ L reaction system₂O。

The reaction conditions are as follows: adopting 2-step PCR reaction conditions: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5 s; annealing at 64 ℃ for 34 s; denaturation at 95 ℃ for 10s, annealing at 65 ℃ for 60s, elongation at 97 ℃ for 1s, and denaturation-annealing-elongation repeated for 38 cycles.

As shown in fig. 1c, (c) RNA was extracted from Md5 infected CEF cells and normal CEF cells. SAA mRNA expression changes were detected by qPCR.

FIG. 1c can demonstrate that infection of CEF cells with a virulent strain of MDV Md5 can activate the expression of SAA in CEF cells early.

Example 2: overexpression of SAA remarkably inhibits replication of MDV virulent strain Md5 virus

In order to clearly activate SAA to inhibit Md5 replication, this example constructed SAA overexpression plasmids, and verified the inhibitory effect of SAA on Md5 viral replication by overexpressing SAA.

1. Effect of overexpression of SAA detection on MDV replication

(1) Constructing eukaryotic expression vector. The construction of SAA eukaryotic expression plasmid is completed by the Gima gene company by adopting the prior art, and the invention selects PEX-3 vector (pGCMV/MCS/Neo) eukaryotic expression plasmid to express SAA.

(2) Then transfecting the constructed SAA eukaryotic expression plasmid into a CEF cell;

1) culturing the cells in a 12-hole plate before transfection to ensure that the cells maintain the optimal concentration and state;

2) and (3) rewarming the reagent: heating the X-tremeGENE HP DNA Transfection Reagent, PEX-SAA eukaryotic expression plasmid or PEX-3 empty vector and diluent to about 20 ℃, and mixing the X-tremeGENE HP DNA Transfection Reagent by short vortex;

3) solution preparation: using Opti-MEM medium as a diluent, the SAA overexpression plasmid was diluted to a final concentration of 1. mu.g/100. mu.L medium and gently mixed. mu.L of the dilution containing 1. mu.g of the SAA-overexpressing plasmid was added separately to sterile centrifuge tubes. The X-tremeGENE HP DNA Transfection Reagent was added directly to the medium containing the diluted DNA, the ratio of plasmid DNA to Transfection Reagent was 1: 3, in the process, the gun head does not contact the wall of the centrifuge tube; the volume of diluent used is more than 100 μ L;

4) and (3) incubation: incubating the transfection complex for 15min at about 20 ℃;

5) transfection: the cells taken out from the incubator do not need to discard the original culture medium, and the transfection compound is directly dripped into the cells;

6) MDV infected cells: when CEF cells were transiently transfected with SAA overexpression plasmid for 6h, the original medium was discarded, washed 3 times with PBS, MDV was inoculated, 1mL of DMEM medium containing Md5 strain was added to each well, and the cells were maintained at 37 ℃ for 36h, and harvested. Extracting cell RNA, cracking cell protein, detecting SAA expression level by qPCR, and detecting MDV replication level by virus titer, qPCR and western blot.

The results are shown in FIGS. 2A-D. (A) After CEF cells are transfected with PEX-SAA or a control plasmid PEX-3, Md5 is inoculated, and after the cells are maintained for 36 hours, cell RNA is extracted. The level of SAA mRNA is detected by qPCR, and the result shows that the expression amount of SAAmRNA in the cells of the SAA overexpression group is obviously higher than that of the control group. (B) The level of the Meq mRNA of the CEF cells after being transfected with PEX-SAA or PEX-3 and inoculated with Md 536 h is detected by qPCR, and the result shows that the expression amount of MDV Meq protein mRNA of the SAA group cells is obviously lower than that of the control group. (C) After CEF cells were transfected with PEX-SAA or the control plasmid PEX-3, MDV was inoculated and maintained for 36 h. The virus titer of MDV in CEF cells of different groups is detected by a virus titer experiment, and the result shows that the virus titer of MDV in the SAA overexpression group is obviously lower than that of a control group. (D) After CEF cells were transfected with PEX-SAA or the control plasmid PEX-3, MDV was inoculated and maintained for 36 h. The expression level of MDV Meq protein in the cells is detected by western blot, and the result shows that the expression level of the Meq protein in the cells of the SAA group is obviously lower than that of the control group.

It is demonstrated by FIGS. 2A-D that overexpression of SAA inhibits replication of the MDV virulent strain Md5 virus compared to the control group. The above results show that overexpression of SAA can significantly inhibit replication of virulent strain Md 5.

Example 3: interference with SAA significantly promotes MDV replication

To clarify the importance of the expression level of SAA on MDV load, this example constructs SAA interfering RNA, and verifies the importance of SAA on host cell resistance to MDV replication by knocking down SAA.

Construction of SAA interference plasmids

(1) Reverse verification is carried out on the result of inhibiting MDV virus replication by SAA through siRNA, and 3 SAA interference RNAs are constructed by Germa company, and the nucleotide sequences of the SAA interference RNAs are shown as follows;

siRNA-1：

sense strand: ccagcgguau caaguuugut t, respectively; (SEQ ID NO.9)

Antisense strand: acaaacuuga uaccgcuggt t are provided. (SEQ ID NO.10)

siRNA-4：

Sense strand: gugcugacaa guauuuccat t, respectively; (SEQ ID NO.11)

Antisense strand: uggaaauacu ugucagcact t are provided. (SEQ ID NO.12)

siRNA-NC：

Sense strand: uucuccgaac gugucacgut t, respectively; (SEQ ID NO.13)

Antisense strand acgugacacg uucggagaat t. (SEQ ID NO.14)

2. Effect of interfering SAA detection on MDV replication

(1) Culturing the cells in a 12-hole plate before transfection to ensure that the cells maintain the optimal concentration and state;

(2) and (3) rewarming the reagent: heating the X-tremeGENE HP DNA Transfection Reagent, the three SAA interfering siRNAs and the diluent to about 20 ℃, and mixing the X-tremeGENE HP DNA Transfection Reagent by short vortex;

(3) solution preparation: using Opti-MEM medium as a diluent, 100. mu.L of the diluent containing 1. mu.g of the SAA interfering plasmid was added to each sterile centrifuge tube. Adding the X-tremeGENE HP DNA Transfection Reagent directly into a culture medium containing diluted DNA, wherein the volume ratio of the plasmid DNA to the Transfection Reagent is 1: 3, in the process, the gun head does not contact the wall of the centrifuge tube; the volume of diluent used is more than 100 μ L;

(4) and (3) incubation: incubating the transfection complex for 15min at about 20 ℃;

(5) transfection: the cells taken out from the incubator do not need to discard the original culture medium, and the transfection compound is directly dripped into the cells;

(6) MDV infected cells: after cells are transiently transfected for 6h, the original culture medium is discarded, the cells are washed 3 times by PBS, and an MDV virulent strain Md5 strain is inoculated and maintained at 37 ℃ for 36 h.

(7) Detection of MDV load: cells were harvested and the amount of MDV virus replication was determined by qPCR, virus titer, and western blot, respectively.

The results are shown in FIGS. 3E-H. (E) Transfection of CEF cells with siRNA (siRNA-1, siRNA-4 or siRNA-NC) followed by inoculation of MDV, the amount of SAA expression was examined by qPCR, and the results showed that the amount of SAA expression in the cells of the siRNA-4 transfected group was lower than that of the control group. (F) After transfection of CEF cells (siRNA-1, siRNA-4 or siRNA-NC) with siRNA, cells were infected with MDV and cellular RNA was extracted. The MDV Meq mRNA expression level is detected by qPCR, and the result shows that the MDV Meq mRNA expression level in the cells of the siRNA-4 transfected group is higher than that of the control group, and the MDV Meq mRNA expression level in the cells of the siRNA-1 transfected group is not different and significant compared with that of the control group. (G) CEF cells were transfected with siRNA-1, siRNA-4 or siRNA-NC and then inoculated with MDV for 36 h. The virus titer of MDV in CEF cells of different groups is detected by a virus titer experiment, and the result shows that the MDV virus titer of the siRNA-4 group is obviously higher than that of a control group. (H) And (3) transfecting cells by using siRNA-1, siRNA-4 or siRNA-NC, then infecting the cells by using MDV, and detecting the MDVMeq protein expression amount by using WB (WB), wherein the MDV Meq protein expression amount of the cells transfected by using the siRNA-4 group is obviously higher than that of a control group. FIGS. 3E-H demonstrate that the knockdown of SAA promotes MDV replication. The above results demonstrate that SAA has the ability to inhibit replication of MDV virulent strain Md 5.

Example 4: obtaining and transformation of recombinant plasmids

Based on the amino acid sequence of SAA, as shown in SEQ ID NO.1, the inventor obtains a nucleotide sequence for coding the amino acid, as shown in SEQ ID NO. 2; on the basis, the inventor constructs pET22b-his-SAA recombinant plasmid (the structural schematic diagram of pET22b-his-SAA recombinant plasmid is shown in FIG. 4) by the Alberson company.

The pET22b-his-SAA recombinant plasmid is transformed into an escherichia coli DH5 alpha competent cell, escherichia coli with positive plasmids is screened out from an LB solid culture medium containing ampicillin, and escherichia coli containing SAA recombinant expression plasmids is screened out through sequencing, wherein the specific transformation steps are as follows:

coli DH 5. alpha. competent cells were thawed on ice, 5. mu.L of pET22b-his-SAA recombinant plasmid was added to 100. mu.L of E.coli DH 5. alpha. competent cells, the contents were mixed by gently rotating the tube, and placed in an ice bath for 30 min. Placing the centrifuge tube in a water bath at 42 ℃ for 60-90 s, then quickly transferring the centrifuge tube to an ice bath for placing for 2-3 min, and paying attention to not shake the centrifuge tube; add 500. mu.L sterile and antibiotic-free LB medium to the centrifuge tube and shake-culture at 37 ℃ for 1 h. And (3) coating a proper amount of transformed competent cells on an Amp-resistant LB plate, carrying out inverted culture at 37 ℃ for 12-16 hours, and picking a single colony for mass culture.

Then extracting the plasmid from the Escherichia coli containing the SAA expression plasmid, transforming the plasmid into Escherichia coli BL21 competent cells, and performing the transformation step according to the steps. Escherichia coli with positive plasmids is screened out from LB solid culture medium containing 100 mug/ml ampicillin, Escherichia coli capable of expressing SAA is screened out, and the residual bacterial liquid is stored in a refrigerator at-80 ℃ for later use.

Example 5: expression, purification and renaturation of SAA

Expression of SAA

A bacterial solution (positive bacterial solution) of escherichia coli capable of expressing SAA was prepared according to Amp: positive bacteria liquid: LB liquid medium was mixed at a ratio of 1:10:1000 (vol.), propagated on a shaker at 37 ℃ and expressed SAA.

The preparation method comprises the following specific steps:

respectively adding 100mL of LB liquid culture medium, 100 mu L of Amp (ampicillin) and 1mL of escherichia coli which is constructed in the embodiment 4 and can express SAA into a 500mL conical flask, and culturing for 6h on a shaking table at 37 ℃ and 220r/min to obtain a bacterial liquid; and (3) taking the precipitate, carrying out heavy suspension on the precipitate by PBS, crushing the precipitate by an ultrasonic crusher for 3s at intervals of 4s, repeating the steps for 99 times to form a cycle, and crushing the thallus until the solution is transparent. Centrifuging at 4 deg.C and 12000r/min for 15min, discarding supernatant, adding PBS to resuspend precipitate, centrifuging by the same method, and cleaning precipitate.

Purification of SAA

Purifying protein by using nickel ion SAA affinity and a chromatographic column, adding sufficient LE Buffer to resuspend and precipitate, placing the mixture in a refrigerator at 4 ℃ overnight to dissolve an inclusion body and denature the protein, centrifuging the inclusion body after overnight dissolution at 4 ℃ at 12000r/min for 5min, discarding the precipitate, adding the solution into the chromatographic column, controlling the flow rate to carry out loading for 3-5 s/drop, collecting effluent, and if necessary, enabling the effluent to be loaded for multiple times to improve the yield.

After the sample loading is finished, washing the chromatographic column by using 2 times of column volume of washing buffer (containing lower concentration of imidazole which can wash away the impurity protein and only retain the target protein), adding 2mL of elution buffer into the chromatographic column, repeatedly loading the effluent eluent for 5-10 times, and finally obtaining the eluent containing the purified SAA, wherein the purification efficiency can be detected by SDS-PAGE.

The various reagents used in the above procedure were as follows:

Lysis(LE Buffer)：100mM Na₂HPO₄、10mM Tris·cl、8M Urea、PH＝8.0；

washing buffer solution: 100mM Na₂HPO₄、10mM Tris·cl、10mM imidazole、8M Urea、PH＝8.0；

Elution buffer: 100mM Na₂HPO₄、10mM Tris·cl、250mM imidazole、8M Urea、PH＝8.0；

3. Detection of SAA after purification (SDS-PAGE)

SDS-PAGE detection of purified SAA, the procedure was as follows:

(1) first, 12% separation glue is prepared, a cleaned glass plate is fixed by a clamp of a base (vertically placed), and distilled water is added to check whether the fixing plate is completely fixed. Then pouring out distilled water, quickly injecting the prepared gel in proportion between two glass plates by using a 1mL liquid transfer gun, and adding a certain amount of distilled water at the upper end of the separation gel for sealing so as to ensure that the separation gel is flat and solidified. After the separation gel is completely solidified within 35min, pouring off the water on the upper layer of the separation gel, and sucking the residual water with filter paper as much as possible. Adding the prepared upper layer concentrated glue, and inserting a comb (1mm) with proper pore size, wherein air bubbles are prevented from generating in the process, and the comb can be used after the upper layer concentrated glue is completely solidified (about 30 min).

(2) The purified SAA was added to 1/5 volumes of 5 Xprotein loading buffer in PE tubes and boiled in distilled water for 5 min.

(3) And pulling out a comb of the concentrated gel, adding the prepared 1 xTris-glycine protein electrophoresis buffer solution into the electrophoresis tank to enable the electrophoresis buffer solution to completely submerge the sample adding hole, loading the sample by using a 10 mu L pipette, and loading the sample by using a 20 mu L/hole. And (4) correctly connecting the anode and the cathode of the electrophoresis tank with the electrophoresis apparatus, and turning on a power supply. And (3) performing electrophoresis by using a voltage of 80V, increasing the voltage to 100V when the sample is concentrated on the concentrated gel to form a straight line and reaches the bottom of the concentrated gel and enters the separation gel, and stopping electrophoresis when the sample reaches the bottom of the separation gel.

(4) Dyeing: after the electrophoresis, the gel was carefully cut with a razor blade, and placed in a petri dish with Coomassie brilliant blue staining solution and stained for 40 min.

(5) And (3) decoloring: after dyeing, placing the gel in a decolorizing solution, slowly shaking on a shaking table, periodically replacing the decolorizing solution for more than 2 times until the blue background of the gel is completely removed, placing the gel in distilled water to stop decolorizing, and photographing for storage.

The purified SAA was examined by SDS-PAGE, and the results are shown in FIG. 5, showing that the protein purification effect was good.

Renaturation of SAA

The method adopts urea with different concentrations to carry out dialysis renaturation on denatured protein, and comprises the following specific steps:

purified SAA is taken out by using a dialysis bag with proper molecular cut-off amount (the molecular weight is about 5KDa) and is respectively placed in renaturation solutions 1-4 (according to the sequence of the renaturation solutions 1-4), each renaturation solution is kept for 3-4h, and finally the SAA is placed in PBS overnight, so that the denatured protein is refolded, the biological activity of the protein is recovered, and the SAA with the biological activity is expressed. And drawing a standard curve by using the BCA protein quantitative detection kit, wherein the concentration of the detected small peptide is 1029 mu g/mL as shown in figure 6, and 2-3mg of SAA small peptide can be finally obtained by calculating bacterial liquid per liter.

The adopted renaturation liquid comprises the following components:

renaturation liquid 1: 500mM Tris. cl, 100mM NaCl, 4M Urea, pH 8.0

Renaturation liquid 2: 500mM Tris. cl, 100mM NaCl, 3M Urea, pH 8.0

Renaturation liquid 3: 500mM Tris. cl, 100mM NaCl, 2M Urea, pH 8.0

Renaturation liquid 4: 500mM Tris. cl, 100mM NaCl, 1M Urea, pH 8.0.

Example 6: recombinant SAA in vitro Marek's virus resistance experiment

1. Proliferation-toxicity assay of recombinant SAA on cells

Prior to performing anti-MDV experiments on recombinant SAA on cells, the cytotoxic effect of an SAA on CEF was examined by Cell Counting kit-8 (CCK-8).

CCCK-8 cell proliferation-toxicity assay protocol:

preparing 100 mu L DF-1 cell suspension in a 96-well plate, and culturing the 96-well plate in a cell culture box for 24 h; sequentially adding 10 mu L of SAA protein with different concentrations into the hole of a 96-well plate (the protein concentration is sequentially 2.5 mu g/mL, 5 mu g/mL and 10 mu g/mL); incubating the 96-well plate in a cell culture box for 24 hours; add 10. mu.L of CCK-8 solution to each well; incubating the 96-well plate in a cell culture box for 4 hours; absorbance at 450nm was measured with a microplate reader, as shown in FIG. 7, SAA application did not produce significant toxicity to CEF cells.

To determine whether SAA inhibits MDV infection in vitro, these 3 different concentrations were tested for MDV vaccination group, MDV + 2.5. mu.g/mL SAA, MDV + 5. mu.g/mL SAA, and MDV + 10. mu.g/mL SAA, respectively.

Design and synthesis of MDV real-time fluorescent quantitative PCR primer

According to the published MDV gene sequence, a real-time fluorescent quantitative PCR primer is synthesized and synthesized by Huada.

The primer sequence of the avian reference gene GPADH is as follows:

F:5’-GAACATCATCCCAGCGTCCA-3’，(SEQ ID NO.5)

R:5’-CGGCAGGTCAGGTCAACAAC-3’。(SEQ ID NO.6)

the sequence of the Meq fluorescent quantitative primer is as follows:

F:5′-TTGTCATGAGCCAGTTTGCCCTAT-3′，(SEQ ID NO.7)

R:5′-TGAGGGAGGTGGAGGAGTGCAAAT-3′。(SEQ ID NO.8)

3. recombinant SAA in vitro anti-MDV real-time fluorescent quantitative PCR detection

And (3) carrying out CEF cell experiments on the purified and renatured recombinant SAA, transferring the CEF cells to a 12-well plate, adding the SAA when the cell density reaches 50-60%, taking the group added with PBS as a Negative Control (NC), inoculating MDV (Md5) to all the groups when the cell density reaches 70-80%, and respectively collecting the cells after maintaining for 24 h. Total cellular RNA was extracted using a total cellular RNA extraction kit (QIAGEN, America), and then the RNA was reverse-transcribed into cDNA. Real-time fluorescent quantitative PCR detection was performed as shown in fig. 8.

The real-time fluorescent quantitative PCR detection result shows that when the concentration of the added SAA is 10 mug/mL, the recombinant SAA in vitro anti-MDV has obvious effect, and meanwhile, the minimum concentration of the recombinant SAA in vitro anti-MDV can be determined to be 5 mug/mL.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> inhibitors of viral replication and uses thereof

<130> 2022

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 127

<212> PRT

<213> SAA protein

<400> 1

Met Arg Leu Cys Ile Cys Phe Val Leu Leu Ser Ile Val Leu Tyr Ala

1 5 10 15

Ser Ala Asp Asn Pro Phe Ser Ser Gly Ile Lys Phe Val Arg Asp Ala

20 25 30

Ala Gly Gly Ala Arg Asp Met Trp Arg Ala Tyr Arg Asp Met Arg Glu

35 40 45

Ala Asn Tyr Ile Gly Ala Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr

50 55 60

Asp Ala Ala Arg Arg Gly Pro Gly Gly Ala Trp Ala Ala Lys Val Ile

65 70 75 80

Ser Asp Ala Arg Glu Gly Trp Gln Ser Arg Val Ser Gly Arg Gly Ala

85 90 95

Glu Asp Thr Arg Leu Asp Gln Glu Ala Asn Glu Trp Gly Arg Arg Gly

100 105 110

Gly Asp Pro Asn Arg Phe Arg Pro Ala Gly Leu Pro Ser Lys Tyr

115 120 125

<210> 2

<211> 386

<212> DNA

<213> SAA protein-encoding gene

<400> 2

ccatgaggct ctgtatctgc ttcgtgttgc tctccattgt tctgtatgca agtgcagaca 60

acccattctc cagcggtatc aagtttgtca gggatgcagc tggaggcgca cgggatatgt 120

ggagagcata ccgggacatg cgtgaggcaa actacattgg tgctgacaag tatttccatg 180

ctcgtggcaa ttacgatgct gctcggagag gacctggagg tgcttgggca gccaaagtga 240

tcagcgatgc ccgggagggc tggcagagca gggtgagcgg cagaggcgcg gaggacaccc 300

ggctcgacca ggaggccaac gagtggggca ggagaggcgg cgatcccaac cgcttcaggc 360

ccgcaggact tcctaacaaa tactga 386

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

ttctccagcg gtatcaagtt 20

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence

<400> 4

ccgagcagca tcgtaatt 18

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

gaacatcatc ccagcgtcca 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

cggcaggtca ggtcaacaac 20

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence

<400> 7

ttgtcatgag ccagtttgcc ctat 24

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence

<400> 8

tgagggaggt ggaggagtgc aaat 24

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<400> 9

ccagcgguau caaguuugut t 21

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

acaaacuuga uaccgcuggt t 21

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<400> 11

gugcugacaa guauuuccat t 21

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<400> 12

uggaaauacu ugucagcact t 21

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence

<400> 13

uucuccgaac gugucacgut t 21

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<400> 14

acgugacacg uucggagaat t 21

Claims

1. The application of serum amyloid A in the following (1) or (2):

A1) protein with an amino acid sequence of SEQ ID NO. 1;

2. Use of a gene encoding serum amyloid A in the following (1) or (2):

(2) preparing the drug for inhibiting the replication of Marek's disease virus.

3. The use according to claim 2, wherein the gene encoding serum amyloid A is a nucleic acid molecule represented by i) or ii) or iii) as follows:

i) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 2;

4. The application of the biological material related to serum amyloid A in the following (1) or (2):

(2) preparing the drug for inhibiting the replication of Marek's disease virus.

5. The use according to claim 4, wherein the biological material associated with serum amyloid A is any one of:

B1) a recombinant expression vector containing a serum amyloid A coding gene;

B2) a recombinant microorganism comprising a gene encoding serum amyloid A;

B3) a transgenic cell line comprising a gene encoding serum amyloid A;

6. A drug for inhibiting replication of Marek's disease virus, which comprises an effective amount of serum amyloid A as an active ingredient.

7. The pharmaceutical according to claim 6, wherein the concentration of serum amyloid A in the pharmaceutical is 5 μ g/mL to 10 μ g/mL.

8. The medicament of claim 6, further comprising: one or more of a substance for enhancing or increasing the activity of serum amyloid A, a substance for increasing the content of serum amyloid A and a substance for increasing the expression level of a gene encoding serum amyloid A.