CN113105537B

CN113105537B - Host protein for promoting replication of influenza A virus and application thereof

Info

Publication number: CN113105537B
Application number: CN202110418462.5A
Authority: CN
Inventors: 刘月月; 黄中利; 殷斌; 吴家强; 李桂明; 林树乾; 杨世发; 赵增成; 祝钰玮
Original assignee: Shandong Yiyuan Pharmaceutical Co ltd; Poultry Research Institute Shandong Academy of Agricultural Sciences
Current assignee: Shandong Yiyuan Pharmaceutical Co ltd; Poultry Research Institute Shandong Academy of Agricultural Sciences
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2022-08-23
Anticipated expiration: 2041-04-19
Also published as: CN113105537A

Abstract

The invention provides a host protein for promoting the replication of influenza A virus, and the amino acid sequence of the host protein is shown as SEQ ID NO. 1. The host protein can be used for proliferating the influenza A virus strain or preparing a medicament for preventing and treating the influenza A virus strain as a target spot, and has important significance in the aspect of developing new medicaments.

Description

Host protein for promoting replication of influenza A virus and application thereof

Technical Field

The invention belongs to the field of molecular biology and immunology, and particularly relates to an application of a host protein immunoglobulin-like receptor ILDR1 in promoting the expression of an influenza virus NP protein and the proliferation of an influenza virus.

Background

Influenza A virus once causes a plurality of outbreaks of influenza in human history, becomes one of main viruses seriously threatening human health, and has important significance for public health safety in prevention and treatment. Because influenza viruses belong to RNA viruses with single negative strand segments, novel viruses can be continuously generated through high mutation rate and gene rearrangement, so that the current vaccine immunization and the drug development taking influenza virus proteins as drug targets are relatively delayed. The emergence of drug-resistant strains of influenza is further accelerated by the extensive use of drugs directed against viral targets. Therefore, the development of new mechanism of action anti-influenza drugs is particularly urgent.

The life cycle of influenza viruses includes adsorption, penetration, peeling of the virus, synthesis of mRNA, synthesis of protein, replication of vRNA, assembly and release of viral particles. The various stages of the replication cycle require the assistance of host proteins. Host factors involved in viral replication are relatively invariant compared to viral proteins, and some of the host factors necessary for viral replication are not critical to the host cell.

The influenza a virus ribonucleoprotein (vRNP) complex is the structural basis for transcription and replication of viral RNA and plays an important role in the process of viral infection of cells. In the early stage of virus infection, the vRNP compound is released into cytoplasm of host cells after endocytosis and membrane removal, and enters into nucleus under the action of nuclear localization signal and transport protein, thus promoting virus transcription and replication. vRNP includes RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP protein), where NP protein is a specific functional protein of influenza virus that can serve as a target for specifically blocking influenza virus replication. It is now found that its entry into the nucleus is regulated by host proteins, and a series of host proteins regulating its entry into the nucleus have been reported. Such as alpha-actin-4, CRM1, UAP56, Hsp40 and MOV10, play an important role in promoting or inhibiting the replication process of viruses. The discovery of new interacting proteins will help provide new targets for the control of influenza viruses.

Disclosure of Invention

Aiming at the problems that the vaccine protection effect is reduced and ineffective easily caused by influenza virus variation, the invention provides a novel protein which can act on influenza virus proliferation, is derived from a host, can interact with influenza A virus, and can promote the proliferation of the influenza A virus.

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

A host protein for promoting the replication of influenza A virus has an amino acid sequence shown in SEQ ID NO. 1.

A nucleotide sequence of the host protein. Preferably, the nucleotide sequence of the host protein is shown in SEQ ID NO. 2.

A recombinant vector, an engineering bacterium and a cell line containing the host protein nucleic acid sequence. The recombinant vector is selected from plasmids. The engineering bacteria are selected from escherichia coli. The cell line is selected from COS7 cell line, HEK293T cell line, A549 cell line and MDCK cell line.

An application of the host protein in the propagation of the influenza A virus strain.

An interfering RNA of the above host protein. The interfering RNA is double-stranded siRNA, and the sequence of the interfering RNA is shown as SEQ ID NO. 3 or SEQ ID NO. 4.

An application of the siRNA in preparing a medicament for preventing and treating influenza A.

The invention has the following advantages:

the host protein provided by the invention can regulate and control the replication of the influenza A virus, can be used for improving the virus amplification amount in a scientific research process, can be used for researching and developing influenza A medicines by taking the host protein as a target, and has important significance in the aspect of new medicine research and development. The interfering RNA provided by the invention can obviously reduce the expression level of host protein for promoting the replication of influenza A virus, thereby obviously inhibiting the replication of influenza A virus.

Drawings

FIG. 1 shows the expression difference of ILDR1 at different times after mice are infected with H1N1 virus;

FIG. 2 shows the H1N1 virus after infection of HEK293T cellsIldr1Change in expression level of (3);

FIG. 3 shows the localization of ILDR1-GFP in COS7 cells by immunofluorescence assay;

FIG. 4 is a Western blot of protein expression by ILDR1-GFP in COS7 cells;

FIG. 5 shows the expression level of H1N1 virus after overexpression of ILDR 1;

FIG. 6 shows the expression level of H1N1 virus after knockdown of ILDR 1;

FIG. 7 shows the expression level of H9N2 virus after overexpression of ILDR 1;

FIG. 8 shows the expression level of H9N2 virus after knockdown of ILDR 1.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.

Example 1 obtaining of ILDR1 Gene

H1N1 strain (isolated from a pig farm with a certain disease in Qingdao Shandong) is infected into 42-day-old C57BL/6J mice, and lungs infected with experimental groups and control groups at different time points are collected for high-throughput sequencing. The bioinformatics analysis finds that: the expression level of ILDR1 was significantly increased compared to the control group. Total protein was extracted from the lungs immediately after 0, 1, 3, 5, 7 and 14 days of infection, respectively, and Western blot analysis was performed, whereby it was found that the level of ILDR1 was significantly increased by 15-fold or more on the first day after infection, the expression level began to decrease on 7 days as the viral load decreased, and the normal level was substantially restored on 14 days (FIG. 1).

HEK293T cells were infected with H1N1 SIV at different MOI (MOI =0, 0.01, 0.1, 1, 10), harvested 24H after infection and detected by qRT-PCRIldr1Change in expression (FIG. 2A). Similarly, HEK293T cells were infected with MOI = 1H 1N1 SIV, harvested 0H, 6H, 12H, 24H, 48H, 72H post infection, and detected using qRT-PCRIldr1Change in expression (FIG. 2B). The results show that ILDR1 mRNA expression following SIV infection is significantly increased in a dose and time dependent manner.

Referring to a mouse genome reference sequence on an NCBI website, designing a cloning primer of a full-length CDS region of a mouse ILDR1 gene according to a transcriptome splicing sequence, introducing Ecor1 enzyme cutting sites and Sal1 enzyme cutting sites (underlined) into an upstream primer and a downstream primer respectively, and leading the sequences to be as follows:

using mouse lung tissue cDNA as a template to carry out PCR amplification, wherein an amplification system is as follows:

the amplification reaction was as follows:

the product of about 1600bp is obtained by amplification, the product is connected to pEGPFN2 plasmid after being recovered by glue, the sequencing is carried out on the positive plasmid (ILDR 1-GFP plasmid), the sequence of the target gene is shown as SEQ ID NO. 2, the total code is 538 amino acids, and the sequence of the amino acids is shown as SEQ ID NO. 1.

Example 2 expression of ILDR1 protein

The COS7 cell line was transfected with the ILDR1-GFP plasmid obtained in example 1 and pEGPFN2 empty vector plasmid not linked to the target gene using Lip2000 liposome, and the group treated with only the transfection reagent and without any plasmid was also used as a negative control group. The transfection efficiency was determined by observing the green fluorescence under the microscope 24h after transfection. As a result, the transfection efficiency of ILDR1-GFP reached 50% or more, as shown in FIG. 3. The above cells transfected with ILDR1-GFP and pEGPFN2 vectors were cultured in CO ₂ Culturing in an incubator for 24h, extracting total protein of cells, and performing Western blot detection by using Anti-GFP tagged antibodies, wherein the result is shown in figure 4, and the ILDR1-GFP can be effectively and highly expressed in COS7 cells.

Example 3 Effect of regulation of ILDR1 protein amount on the proliferation of influenza A Virus

1. Over-expressionIldr1Promoting influenza virus replication

HEK with good growth state293T cells at 2X 10 ⁵ Inoculating into 6-well plate at density of one/mL, transfecting ILDR1-GFP and pEGPFN2 vector the next day, infecting virus with MOI =0.1 24h later, collecting cell supernatant 24h later after inoculation, and performing TCID ₅₀ And viral RNA detection. The results are shown in FIG. 5: overexpression compared to negative control groupIldr1Rear end，The expression level of influenza virus is increased.

2. Knock-downIldr1Inhibiting influenza virus replication

According to the general principle of si-RNA design, siRNA targets were designed and the following sequences were synthesized:

the si-ILDR1 and NC described above were transfected into HEK293T cells in CO ₂ After culturing for 48h in an incubator, extracting total RNA, and detecting the knockout efficiency by using Q-PCR. The results show that: both pairs of si-RNAs can be knocked downIldr1Above 50%.

HEK293T cells in good growth state were cultured at 2X 10 ⁵ one/mL density was inoculated in 6-well plates, si-ILDR1 and NC were transfected the next day, virus with MOI =0.1 was infected 24h later, cell supernatants were collected 48h later for virus RNA detection. The results are shown in FIG. 6: knockdown compared to negative control group (NC group)Ildr1Thereafter, replication of influenza virus can be inhibited.

Example 4 Effect of modulation of ILDR1 protein amount on the proliferation of H9N2 influenza Virus

Referring to the method in example 3, HEK293T cells with good growth status were transfected with ILDR1-GFP and pegfn 2 vectors, infected with H9N2 virus with MOI =0.1 24H later, and cell supernatants were collected 24H later for virus RNA detection. The results are shown in FIG. 7: overexpression compared to negative control groupIldr1Rear end，The expression level of the H9N2 influenza virus is obviously increased by more than 6 times. si-ILDR1 and NC were transfected 24h before infection with virus with MOI =0.1, cell supernatants were collected 48h after inoculation, and viral RNA was detected. The results are shown in FIG. 8: knockdown compared to negative control group (NC group)Ildr1After that, the replication of the H9N2 influenza virus can be obviously inhibited.

Sequence listing

<110> poultry institute of academy of agricultural sciences of Shandong province

<120> host protein for promoting replication of influenza A virus and application thereof

<130> 20210409

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 537

<212> PRT

<213> Mus musculus

<400> 1

Met Gly Cys Gly Leu Leu Ala Ala Gly Leu Leu Leu Phe Thr Trp Leu

1 5 10 15

Pro Ala Gly Cys Leu Ser Leu Leu Val Thr Val Gln His Thr Glu Arg

20 25 30

Tyr Val Thr Leu Phe Ala Ser Val Thr Leu Lys Cys Asp Tyr Thr Thr

35 40 45

Ser Ala Gln Leu Gln Asp Val Val Val Thr Trp Arg Phe Lys Ser Phe

50 55 60

Cys Lys Asp Pro Ile Phe Asp Tyr Phe Ser Ala Ser Tyr Gln Ala Ala

65 70 75 80

Leu Ser Leu Gly Gln Asp Pro Ser Asn Asp Cys Ser Asp Asn Gln Arg

85 90 95

Glu Val Arg Ile Val Ala Gln Arg Arg Gly Gln Ser Glu Pro Val Leu

100 105 110

Gly Val Asp Tyr Arg Gln Arg Lys Ile Thr Ile Gln Asn Arg Ala Asp

115 120 125

Leu Val Ile Asn Glu Val Met Trp Trp Asp His Gly Val Tyr Tyr Cys

130 135 140

Thr Ile Glu Ala Pro Gly Asp Thr Ser Gly Asp Pro Asp Lys Glu Val

145 150 155 160

Lys Leu Ile Val Leu His Trp Leu Thr Val Ile Phe Ile Ile Leu Gly

165 170 175

Ala Leu Leu Leu Leu Leu Leu Ile Gly Val Cys Trp Cys Gln Cys Cys

180 185 190

Pro Gln Tyr Cys Cys Cys Tyr Ile Arg Cys Pro Cys Cys Pro Thr Arg

195 200 205

Cys Cys Cys Pro Glu Glu Ala Leu Ala Arg His Arg Tyr Met Lys Gln

210 215 220

Val Gln Ala Leu Gly Pro Gln Met Met Glu Lys Pro Leu Tyr Trp Gly

225 230 235 240

Ala Asp Arg Ser Ser Gln Val Ser Ser Tyr Ala Met Asn Pro Leu Leu

245 250 255

Gln Arg Asp Leu Ser Leu Gln Ser Ser Leu Pro Gln Met Pro Met Thr

260 265 270

Gln Met Ala Ala His Pro Pro Val Ala Asn Gly Val Leu Glu Tyr Leu

275 280 285

Glu Lys Glu Leu Arg Asn Leu Asn Pro Ala Gln Pro Leu Pro Ala Asp

290 295 300

Leu Arg Ala Lys Ser Gly His Pro Cys Ser Met Leu Ser Ser Leu Gly

305 310 315 320

Ser Ala Glu Val Val Glu Arg Arg Val Ile His Leu Pro Pro Leu Ile

325 330 335

Arg Asp Pro Pro Ser Ser Arg Thr Ser Asn Pro Ser His Gln Gln Arg

340 345 350

Leu Asn Ala Val Ser Ser Arg His Cys Asp Leu Ser Glu Arg Pro Arg

355 360 365

Gln Arg His His Ser Asp Phe Leu Arg Glu Leu Gln Asp Gln Gly Met

370 375 380

Arg Pro Trp Ala Pro Gly Arg Gly Glu Leu Asp Pro His Trp Ser Gly

385 390 395 400

Arg His His Arg Ser Arg Pro Ser Glu Ser Ser Met Pro Trp Ser Asp

405 410 415

Trp Asp Ser Leu Ser Glu Cys Pro Ser Ser Ser Glu Ala Pro Trp Pro

420 425 430

Pro Arg Arg Pro Glu Pro Arg Glu Gly Ala Gln Arg Arg Glu Arg Arg

435 440 445

Arg His Arg Ser Tyr Ser Pro Pro Leu Pro Ser Gly Pro Ser Ser Trp

450 455 460

Ser Ser Glu Glu Glu Lys Glu Ser Leu Pro Arg Asn Trp Gly Ala Gln

465 470 475 480

Arg Arg His His His Arg Arg Arg Arg Ser Gln Ser Pro Asn Trp Pro

485 490 495

Glu Glu Lys Pro Pro Ser Tyr Arg Ser Leu Asp Val Thr Pro Gly Lys

500 505 510

Asn Asn Arg Lys Lys Gly Asn Val Glu Arg Arg Leu Glu Arg Glu Ser

515 520 525

Ser His Ser Gly Arg Ser Val Val Ile

530 535

<210> 2

<211> 1614

<212> DNA

<213> Mus musculus

<400> 2

atgggctgcg gattgctcgc tgctggcctg ctcctcttca cctggctccc agcagggtgt 60

ctgtccttgc tagtcacagt ccagcacaca gaacgctatg ttactctgtt tgcctccgtt 120

accctcaagt gtgactacac cacctctgcc cagctccagg acgtggttgt gacatggcgc 180

ttcaagtcct tctgcaagga tcccatcttt gactacttct ctgcctcata ccaggcagct 240

ttgtccctgg gccaggaccc ctccaatgac tgtagtgaca atcagaggga agttcgcatc 300

gtggcgcagc ggcgtgggca gagtgagccc gtgctggggg tggattaccg gcaacgcaag 360

atcaccatcc agaaccgagc agatcttgtg attaatgaag tgatgtggtg ggatcatgga 420

gtatactatt gtaccatcga ggctccagga gacacgtcag gagacccaga taaggaggtg 480

aagctcattg tcctgcattg gctgacagtg attttcatca ttcttggagc cctcctactc 540

ctgctgctga ttggtgtatg ctggtgccag tgttgtccgc agtattgctg ctgctatatc 600

cgctgcccct gctgtcctac ccgctgttgc tgccctgagg aagccctggc ccgccaccgc 660

tacatgaagc aggttcaggc cctaggtcct cagatgatgg aaaaacccct gtactggggg 720

gcggacagga gctcccaagt ttcatcttat gcaatgaacc cgctgctgca gcgagatctg 780

tccttacagt ccagccttcc acagatgcca atgacccaga tggctgctca ccctccggtg 840

gctaatggtg tcctggaata tttggagaaa gaattgcgga acctcaaccc agcccaacct 900

ctgcctgcgg atctcagagc caaatctggc cacccttgca gcatgctctc ctccctgggc 960

tccgcagagg ttgtggaacg cagagtcatc cacctgcccc cactaatcag agacccaccg 1020

tcctccagga ccagcaaccc ctcacaccag cagcggctca atgctgtttc ttccagacac 1080

tgcgatctga gtgagcgccc gaggcagcgc catcactccg atttcctccg agagctccag 1140

gaccagggga tgagaccctg ggccccgggg agaggggagc tggaccccca ttggagtggg 1200

agacaccacc gctctaggcc cagcgagtca tccatgcctt ggtcagactg ggacagcctg 1260

agcgaatgtc cctcatccag tgaggctcct tggcccccca gacgaccaga gcccagggaa 1320

ggcgcccaga gacgtgagag acgcaggcat cgcagctact cgcctcctct accctcgggc 1380

cccagctctt ggagctctga agaggagaaa gagtcgctgc ccaggaactg gggtgcccag 1440

cgacgtcacc atcaccgccg ccgccgctca cagtctccaa actggcctga ggagaagccg 1500

cccagctacc gctcactgga tgtgactcca ggcaagaaca acaggaaaaa agggaatgtg 1560

gagaggcgct tggagagaga gagctcccat agtggacgga gtgtggtcat ttag 1614

<210> 3

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> si-ILDR1-1

<400> 3

cugcaaggac ccuaucuuut taaagauagg guccuugcag tt 42

<210> 4

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> si-ILDR1-2

<400> 4

gaguuggacc caucguggat tuccacgaug gguccaacuc tt 42

<210> 5

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> NC

<400> 5

uucuccgaac gugucacgut tacgugacac guucggagaa tt 42

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> 1F

<400> 6

aatgaattca tgggctgcgg attgctc 27

<210> 7

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> 1R

<400> 7

attccgtcga ctaatgacca ctccgtcc 28

Claims

1. The application of a host protein in propagating influenza A virus strains is characterized in that the amino acid sequence of the host protein is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the nucleotide sequence of the host protein is as shown in SEQ ID NO 2.

3. The application of a host protein in preparing a medicament for treating influenza A is characterized in that interfering RNA of the host protein is prepared into the medicament, and the amino acid sequence of the host protein is shown as SEQ ID NO. 1.

4. The use of claim 3, wherein the interfering RNA is a double-stranded siRNA having the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.