CN109206501B

CN109206501B - Cat interferon omega, coding gene thereof and application of cat interferon omega in antiviral aspect

Info

Publication number: CN109206501B
Application number: CN201811125211.2A
Authority: CN
Inventors: 徐义刚; 范文禄; 王丽; 王紫微; 朱妍
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2022-03-04
Anticipated expiration: 2038-09-26
Also published as: CN109206501A

Abstract

本发明公开了一种猫干扰素ω、其编码基因及其在抗病毒方面的应用。所述的猫干扰素ω的氨基酸序列如SEQ ID NO.2所示。优选的编码所述的猫干扰素ω的核苷酸序列如SEQ ID NO.1所示。此外，本发明还公开了一种制备所述的猫干扰素ω的方法及其在制备抗病毒药物中的应用。本发明采用病毒刺激(猫肠道冠状病毒)结合干扰素诱生剂刺激(聚肌胞poly I:C)方法对宠物猫体进行刺激，进而诱导猫干扰素基因表达。然后通过PCR扩增得到了一条猫干扰素ω基因，细胞试验显示本发明获得的一种猫干扰素ω具有明显的抗病毒活性，并优于现有已知猫干扰素。本发明的提出为猫干扰素制品的进一步研发和应用奠定了基础。The invention discloses a cat interferon ω, its encoding gene and its application in anti-virus. The amino acid sequence of the cat interferon ω is shown in SEQ ID NO.2. The preferred nucleotide sequence encoding the feline interferon ω is shown in SEQ ID NO.1. In addition, the present invention also discloses a method for preparing the feline interferon ω and its application in preparing an antiviral drug. The present invention uses virus stimulation (feline intestinal coronavirus) combined with interferon inducer stimulation (poly myocytopoly I:C) method to stimulate the pet cat body, thereby inducing cat interferon gene expression. Then a feline interferon ω gene is obtained by PCR amplification, and the cell test shows that the feline interferon ω obtained by the present invention has obvious antiviral activity, and is superior to the existing known feline interferon. The proposal of the present invention lays a foundation for the further development and application of feline interferon products.

Description

Cat interferon omega, coding gene thereof and application of cat interferon omega in antiviral aspect

Technical Field

The invention relates to a novel cat interferon omega, a coding gene thereof and application thereof in the aspect of antivirus. The invention belongs to the technical field of veterinary drugs.

Background

Interferon is an antiviral cytokine produced when the body is infected by virus, is released by infected cells to inhibit the proliferation of virus, and has broad-spectrum antiviral and immune response enhancing effects. There are two broad classes of interferons that have been identified: form I and form II. The type I interferon mainly comprises IFN-alpha, IFN-beta and IFN-omega; the type II interferon is mainly IFN-gamma. At present, the research of human interferon is rapidly progressed, for example, the recombinant human interferon alpha in genetic engineering has been widely used for the treatment of human virus infection, and the recombinant human interferon omega has also been used for the treatment of SARS.

In recent years, the breeding quantity of pet cats in China is rapidly increased, and the pet cats become a part of leisure life of people, and a plurality of virus diseases seriously harm the health and survival of the pet cats. Interferon is the most effective therapeutic drug for treating viral infectious diseases, but the research on cat interferon and the development of clinical preparations are relatively lagged, and the published cat interferon sequence is relatively limited.

The invention discloses a novel cat interferon omega and a coding gene sequence thereof, prepares the novel cat interferon by a gene engineering method and performs antiviral activity determination, and provides a material basis for the research and development of cat interferon biological products.

Disclosure of Invention

The invention aims to provide a novel cat interferon omega and a coding gene thereof.

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

the invention adopts a method of virus stimulation (feline enterocoronavirus) and interferon inducer stimulation (poly I: C) to stimulate pet cats so as to induce the expression of feline interferon genes. Then, according to a cat interferon omega gene sequence published in GenBank, gene sequence comparison is carried out through DNAMAN, a pair of degenerate primers are designed by using Oligo6 to amplify the cat interferon omega gene, and finally, the invention obtains a novel gene sequence for coding the cat interferon omega, and through sequencing and BLAST comparison, the homology of the gene sequence with the published cat interferon omega gene is higher, the gene sequence belongs to a new member of the cat interferon omega, the nucleotide sequence is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The novel cat interferon is prepared by a gene engineering method and antiviral activity determination is carried out on the novel cat interferon, and the result shows that the novel cat interferon omega has good antiviral activity, the effective antiviral concentration is 0.70mg/mL, the novel cat interferon omega is superior to the known cat interferon, and good application potential is shown.

On the basis of the research, the invention provides a novel cat interferon omega, and the amino acid sequence of the cat interferon omega is shown as SEQ ID No. 2.

The nucleotide sequence for coding the feline interferon omega, the recombinant expression vector containing the nucleotide sequence and the host cell containing the recombinant expression vector are also within the scope of the invention. Wherein, preferably, the nucleotide sequence is shown as SEQ ID NO. 1.

Furthermore, the invention also provides application of the feline interferon omega in preparation of antiviral drugs.

Preferably, the medicament is used for treating or preventing viral infectious diseases of cats.

Preferably, the virus includes feline enterocoronavirus and feline parvovirus.

Furthermore, the invention also provides a method for preparing the cat interferon omega, which comprises the following steps:

(1) obtaining a nucleotide sequence for coding the cat interferon omega, and connecting Kpn I and BamH I enzyme cutting sites at two ends of the sequence;

(2) construction of recombinant expression vector pCold-TF-omega

Respectively carrying out double enzyme digestion treatment on an expression vector pCold-TF and the sequence obtained in the step (1) by using restriction enzymes Kpn I and BamH I, recovering a target gene fragment by using a DNA gel recovery kit, and further connecting the sequence obtained in the step (1) into the expression vector pCold-TF by using T4 ligase to construct a recombinant expression vector named pCold-TF-omega;

(3) construction of recombinant bacterium pCold-TF-omega/BL 21

Transforming BL21 competent cells by using the recombinant expression vector pCold-TF-omega, coating the competent cells on an LB agar plate containing ampicillin resistance for culture, and selecting a positive recombinant bacterium named pCold-TF-omega/BL 21;

(4) inducible expression

Overnight plating and activating recombinant bacteria pCold-TF-omega/BL 21, selecting a single colony, inoculating the single colony in an LB liquid culture medium containing 100 mu g/ml ampicillin, and performing shake culture at 37 ℃ overnight; transferring the new culture broth into LB liquid culture medium containing ampicillin resistance according to the volume ratio of 1:10, and culturing at 37 deg.C to OD₆₀₀When the concentration is 0.4-0.6, adding IPTG with the final concentration of 1mmol/L, and inducing for 6h at 37 ℃; and (3) taking the induced recombinant bacterial liquid, centrifuging, discarding the supernatant, adding PBS (phosphate buffer solution) to resuspend the thallus, carrying out ultrasonic treatment, then adding 5 xSDS (sodium dodecyl sulfate) sample lysate, carrying out boiling water bath for 10min, centrifuging, taking the supernatant, carrying out His-tag affinity purification on the recombinant fusion protein, carrying out HRV 3C protease treatment and His-tag affinity purification, and finally obtaining the cat interferon omega.

Wherein, preferably, the nucleotide sequence for coding the feline interferon omega is shown in SEQ ID NO. 1.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention discloses a novel cat interferon omega and a coding gene sequence thereof, and a gene engineering method is used for preparing the cat interferon omega, thereby meeting the requirement on cat interferon in the market.

2. Cell tests show that the novel cat interferon omega disclosed by the invention has obvious antiviral activity and is superior to the existing known cat interferon. The invention lays a foundation for further research and development and application of the cat interferon product.

Drawings

FIG. 1 shows the result of PCR amplification;

m: DNA Marker DL 2000; 1: PCR products; 2: negative control;

figure 2 is the tertiary structure of novel catinterferon omega;

FIG. 3 shows the result of identifying recombinant expression vector pCold-TF-omega;

A. enzyme digestion identification result, M: DNA Marker 8000; 1: the result of the Kpn I-BamH I double enzyme digestion identification of the recombinant plasmid pCold-TF-omega; 2: negative control, b.pcr assay result, M: DNA Marker 8000; 1: PCR amplification result of the recombinant plasmid pCold-TF-omega; 2: negative control;

FIG. 4 is the induced expression profile of feline interferon omega;

A. soluble expression of recombinant protein: m. protein standard molecular weight; 1-5, inducing the recombinant bacteria pCold-TF-omega/BL 21; 6. the non-induced recombinant bacterium pCold-TF-omega/BL 21; 7. the result of induction of the empty vector bacterium pCold-TF/BL 21; B. his-tag affinity purification results of feline interferon omega: m. protein standard molecular weight; 1. the invention relates to a cat interferon omega protein.

Figure 5 is a graph of the potent antiviral activity of feline interferon omega of the invention;

figure 6 is the potent antiviral activity of feline interferon alpha;

figure 7 is the potent antiviral activity of feline interferon omega 2;

figure 8 is the potent antiviral activity of feline interferon omega 9.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.

Example 1 acquisition of novel catinterferon omega

1. Primer design and sequence analysis

According to a cat interferon omega gene sequence published in GenBank, gene sequence alignment is carried out through DNAMAN, a pair of degenerate primers are designed by using Oligo6 to amplify the full length of the cat interferon omega gene, and BamH I and Kpn I restriction enzyme sites (used for constructing a recombinant expression vector for expressing cat interferon) are introduced at the 5 'end and the 3' end of the primers. The primer sequences are shown in Table 1, and the primers were synthesized by Jilin province Cumei Biotech Co., Ltd.

TABLE 1 primer sequences

2. Extraction of feline genomic RNA

The invention adopts a method of virus stimulation (feline enterocoronavirus) and interferon inducer stimulation (poly I: C) to stimulate pet cats so as to induce the expression of feline interferon genes. The specific operation is as follows:

cat kidney cells F81 were seeded in 96-well cell plates and incubated at 37 ℃ with 5% CO₂Culturing in an incubator until cells grow into a monolayer, inoculating feline enteric coronavirus, adsorbing at 37 deg.C for 1 hr, adding a maintenance solution, placing in an incubator at 37 deg.C and 5% CO2, culturing, observing cytopathic condition day by day, freezing and thawing cells until cytopathic Condition (CPE) reaches above 85%, centrifuging at 3000r/min for 10min to obtain virus solution, and determining virus TCID₅₀. Get 100TCID₅₀The virus solution is mixed with 0.5mg of poly I: C, and injected into pet cats for 3 days and 1 time per day. On day 5, euthanizing the cat, obtaining the spleen of the cat under an aseptic condition, cutting the spleen into pieces, freezing and grinding the pieces into powder by liquid nitrogen, and transferring the powder into a centrifuge tube; adding 2mL Trizol to lyse the cells, sucking 800. mu.L of lysed cell lysate and placing it in a 1.5mL centrifuge tube, adding 1/5 volumes of pre-cooled chloroform, shaking and mixingHomogenizing for 15s, centrifuging at 12000rpm and 4 deg.C for 15 min; collecting supernatant, adding equal volume of precooled isopropanol, acting at 4 deg.C for 10min, centrifuging at 12000rpm and 4 deg.C for 10 min; discarding the supernatant, and centrifuging for 10min at 4 ℃ by precooling 75% ethanol at 12000 rpm; the supernatant was discarded, dried at room temperature, and 30. mu.L of deionized water was added to dissolve the precipitate, thereby obtaining total RNA. Then, the extracted total RNA was reverse-transcribed into cDNA using Superscript reverse transcriptase reagent kit, and stored in a freezer at-80 ℃ for use.

3. PCR amplification

And (3) amplifying the cat interferon gene by using the obtained cDNA as a template.

The PCR reaction system is as follows: 10 XPCR Buffer 2.5. mu. L, dNTPs 3. mu.L, Taq DNA polymerase 0.5. mu.L, Primer pair (Primer-F and Primer-R, 10uM) 2. mu. L, cDNA template 5. mu. L, ddH2O 12. mu.L.

And (3) PCR reaction conditions: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 55 ℃ for 45s and 72 ℃ for 30 s; final extension at 72 ℃ for 10 min.

The PCR product was detected by electrophoresis on a 1% agarose gel, and the results are shown in FIG. 1. The PCR product is recovered by a DNA gel recovery kit, sequenced and subjected to BLAST comparison of the sequence, the result is shown in Table 2, the gene sequence obtained by PCR amplification has higher homology with the published gene sequence of the feline interferon omega, belongs to a new member of the feline interferon omega, the nucleotide sequence of the gene sequence is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 2.

TABLE 2 alignment of the gene sequences of the invention with known feline interferon gene homology BLAST

4. Interferon tertiary structure prediction

The protein tertiary structure of the interferon of the present invention was predicted using SWISS-MODEL software, and the prediction results are shown in FIG. 2.

Example 2 Mass expression of novel Cat Interferon omega

1. Construction of recombinant expression vector pCold-TF-omega

The expression vector pCold-TF and the PCR product of the novel cat interferon omega obtained in the embodiment 1 are subjected to double enzyme digestion treatment by restriction enzymes Kpn I and BamH I, a DNA gel recovery kit is adopted to recover a target gene fragment, and then the PCR product of the novel cat interferon omega coding gene is connected into the expression vector pCold-TF by T4 ligase to construct a recombinant expression vector pCold-TF-omega.

2. Construction of recombinant bacterium pCold-TF-omega/BL 21

The recombinant expression vector is transformed into BL21 competent cells, the competent cells are spread on an LB agar plate containing ampicillin resistance for culture, positive recombinant bacteria are selected and identified by adopting an enzyme cutting method (the result is shown in figure 3A) and a PCR method (the result is shown in figure 3B), and the recombinant bacteria are named as pCold-TF-omega/BL 21.

3. Inducible expression

The recombinant bacteria pCold-TF-omega/BL 21 were activated by plating overnight, and a single colony was inoculated into 5mL of LB liquid medium containing 100. mu.g/mL ampicillin and cultured overnight with shaking at 37 ℃. Transferring the new culture broth into 20mL LB liquid medium containing benzyl resistance at a ratio of 1:10, and culturing at 37 deg.C to OD₆₀₀At about 0.5, IPTG was added to a final concentration of 1mmol/L and induction was carried out at 37 ℃ for 6 h. 2mL of the induced recombinant bacterial liquid is taken, centrifuged at 12000rpm for 5min, the supernatant is discarded, 400 mu L of PBS is added to resuspend the thallus and is subjected to ultrasonic treatment, then 100 mu L of 5 xSDS sample lysate is added, boiled water bath is carried out for 10min, centrifuged at 12000rpm for 10min, the supernatant is taken and is subjected to 12% SDS-PAGE analysis, and the result shows that the recombinant protein obtains prokaryotic soluble expression (see figure 4A). And (3) subjecting the recombinant fusion protein (the tag protein and the target protein) to His-tag affinity purification, and then subjecting the recombinant fusion protein to HRV 3C protease treatment and His-tag affinity purification to finally obtain the cat interferon omega protein (shown in figure 4B).

Example 3 determination of antiviral Activity of novel Cat Interferon omega

The antiviral activity of the novel feline interferon omega obtained by the invention is measured by taking feline enterocoronavirus and feline parvovirus as model viruses and adopting a microcytopathic inhibition method. The specific operation is as follows:

cat kidney cells F81 were seeded in 96-well cell plates and incubated at 37 ℃ with 5% CO₂Culturing in an incubator until cell monolayer is reached, and respectively adding 2 times of dilutionThe novel cat interferon omega of the invention acts for 16h, and then 100TCID is added into each hole₅₀The virus liquid of (2) is simultaneously provided with a negative control group and known cat alpha, omega 2 and omega 9 interferon positive control groups, and the cytopathic condition is observed after 24 hours.

TABLE 3 comparison of the antiviral Activity of Interferon

Note: + indicates no cytopathy (i.e. antiviral activity) and-indicates cytopathy (i.e. no antiviral activity).

The results of the antiviral activity test are shown in table 3 and fig. 5-8, and it can be seen from the results that the novel feline interferon omega of the present invention has good antiviral activity, the effective antiviral concentration is 0.70mg/mL (shown in fig. 5), and the activity is superior to the activity of the known feline interferon alpha (0.93mg/mL) (shown in fig. 6), the known feline interferon omega 2 (0.87mg/mL) (shown in fig. 7) and the known feline interferon omega 9 (0.91mg/mL) (shown in fig. 8), and the activity shows good application potential.

Sequence listing

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ttgggtcaga tgagaagatt gtccccattc ttgggtttga gagccaggaa ggacttcaga 180

ttccctagag agatgttgga gggtggtcaa ctgcgtgaag ctcaagctgc tgctgccgtg 240

ttgagagagt tgttgcagca aaccttcaac ctgttgcata ctgagagatc ctccgctgct 300

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Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Ala Leu Leu Thr Cys

1 5 10 15

Arg Pro Gly Gly Ser Leu Gly Cys Ala Leu Pro Gly Tyr His Ala Gln

20 25 30

Val Ser Arg Asp Asn Leu Val Leu Leu Gly Gln Met Arg Arg Leu Ser

35 40 45

Pro Phe Leu Gly Leu Arg Ala Arg Lys Asp Phe Arg Phe Pro Arg Glu

50 55 60

Met Leu Glu Gly Gly Gln Leu Arg Glu Ala Gln Ala Ala Ala Ala Val

65 70 75 80

Leu Arg Glu Leu Leu Gln Gln Thr Phe Asn Leu Leu His Thr Glu Arg

85 90 95

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

100 105 110

Leu His Arg Gln Leu Glu Ala Leu Asp Ala Cys Leu Leu Gln Ala Thr

115 120 125

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Ala Leu Ala Ile

130 135 140

Lys Arg Tyr Phe Gln Ala Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

145 150 155 160

Ser Asp Cys Ala Trp Glu Ile Val Arg Pro Glu Met Met Arg Ala Leu

165 170 175

Ser Ser Ser Ala Thr Leu Gln Asp Ser Leu Ala Ile Lys Asp Gly Asp

180 185 190

Leu Ala Ser Ser

195

Claims

1. The cat interferon omega is characterized in that the amino acid sequence of the cat interferon omega is shown as SEQ ID No. 2.

2. A nucleotide sequence encoding the feline interferon omega of claim 1.

3. The nucleotide sequence of claim 2, wherein the nucleotide sequence is set forth in SEQ ID No. 1.

4. A recombinant expression vector comprising the nucleotide sequence of claim 2 or 3.

5. A host cell comprising the recombinant expression vector of claim 4.

6. The use of feline interferon omega of claim 1 in the preparation of an antiviral medicament for treating or preventing a viral infectious disease in a feline.

7. The use of claim 6, wherein the virus comprises feline enterocoronavirus and feline parvovirus.

8. A method of making the feline interferon omega of claim 1, comprising the steps of:

(1) obtaining a nucleotide sequence encoding the feline interferon omega of claim 1, and ligating Kpn I and BamH I cleavage sites at both ends of the sequence;

(2) construction of recombinant expression vector pCold-TF-omega

(3) construction of recombinant bacterium pCold-TF-omega/BL 21

(4) inducible expression

Overnight plating and activating recombinant bacteria pCold-TF-omega/BL 21, selecting a single colony, inoculating the single colony in an LB liquid culture medium containing 100 mu g/ml ampicillin, and performing shake culture at 37 ℃ overnight; transferring the new culture broth into LB liquid culture medium containing ampicillin resistance according to the volume ratio of 1:10, and culturing at 37 deg.C to OD₆₀₀When the concentration is 0.4-0.6, adding IPTG with the final concentration of 1mmol/L, and inducing for 6h at 37 ℃; get and induceCentrifuging the recombinant bacterial liquid, discarding the supernatant, adding PBS (phosphate buffer solution) to resuspend the thallus, carrying out ultrasonic treatment, then adding 5 xSDS (sodium dodecyl sulfate) sample lysate, carrying out boiling water bath for 10min, centrifuging, taking the supernatant, carrying out His-tag affinity purification on the recombinant fusion protein, carrying out HRV 3C protease treatment and His-tag affinity purification, and finally obtaining the feline interferon omega.

9. The method of claim 8, wherein the nucleotide sequence encoding the feline interferon omega of claim 1 is set forth in SEQ ID No. 1.