PEPTIDE NUCLEIC ACID OF SUBGROUP J AVIAN LEUKOSIS VIRUS AND USES THEREOF

Abstract

A peptide nucleic acid of subgroup J avian leukosis virus and uses of the same are provided. The sequence of the peptide nucleic acid is one or more selected from the following sequences: sequence 1: 5AGACUAAGGCAAAAAUCUGUU-3; sequence 2: 5-ACGACUUAUUGAAAAACUCUC-3; sequence 3: 5-UAUAACCGUCUGUAGUUGGAC-3; sequence 4: 5-ACAUAUUUGAUUAUCUCUCCU-3. The peptide nucleic acid, disclosed in the present invention, can specifically and directly inhibit PRRSV replication, has good antiviral effect and no drug residues, without any toxic side effect and drug resistance.

Claims

1. A peptide nucleic acid, consisting of one or more peptide nucleic acids selected from: a) a nucleic acid sequence of Sequence 1 as shown in the Sequencing List of: TABLE-US-00014 (SEQIDNO:1) 5-AGACUAAGGCAAAAAUCUGUU-3; b) a nucleic acid sequence of Sequence 2 as shown in the Sequencing List of: TABLE-US-00015 (SEQIDNO:2) 5-ACGACUUAUUGAAAAACUCUC-3; c) a nucleic acid sequence of Sequence 3 as shown in the Sequencing List of: TABLE-US-00016 (SEQIDNO:3) 5-UAUAACCGUCUGUAGUUGGAC-3; and d) a nucleic acid sequence of Sequence 4 as shown in the Sequencing List of: TABLE-US-00017 (SEQIDNO:4) 5-ACAUAUUUGAUUAUCUCUCCU-3.

2. The peptide nucleic acid according to claim 1, wherein the peptide nucleic acid is a peptide nucleic acid modified with chitosan.

3. (canceled)

4. The peptide nucleic acid according to claim 1 is an active ingredient in a peptide nucleic acid formulation.

5. The peptide nucleic acid according to claim 4, wherein the peptide nucleic acid formulation is a colon specific controlled-release microcapsule formulation, injectable lyophilized formulation or water-soluble granules for oral use.

6. The peptide nucleic acid according to claim 4, wherein the peptide nucleic acid formulation, further comprises a pharmaceutically acceptable carrier or excipient.

7. The peptide nucleic acid according to claim 1 is used in a preparation of an anti-avian leukosis virus subgroup J (ALV-J) drug.

8. The peptide nucleic acid according to claim 5, wherein the peptide nucleic acid formulation comprises a pharmaceutically acceptable carrier or excipient.

Description

DETAILED DESCRIPTION

[0036] Avian leukosis subgroup J is an infectious disease caused by avian leukosis virus subgroup J (ALV-J) that mainly occurs to chickens, and is characterized by malignant proliferation of hematopoietic cells in chickens. In recent years, the disease has a rapidly rising virus positive rate in the flocks in China and exhibits an expanded host range. The vertical and horizontal transmission of the virus leads to a clinical and subclinical infection, thus causing a high economic loss. By far, there are no effective measures for controlling the disease, and the development of new technologies for preventing and treating ALV-J infection becomes particularly urgent. Furthermore, ALV-J invades the immune system of the chickens, such that the infected flocks are low in immunity, induced immunosuppression, and easily secondarily attacked by other infectious diseases once infected. Up to now, no effective vaccines for preventing the disease are successfully developed. In the present invention, the peptide nucleic acid technology and the antisense nucleic acid technology are combined initially, and used for preventing and treating related diseases caused by ALV-J infection.

[0037] For this purpose, the following technical solutions are provided in the present invention.

[0038] ALV-J strain: strain NS-X11, available from Nansen Central Laboratory of Veterinary Diagnostic techniques Research.

[0039] CEF cells: chick embryo fibroblasts (CEFs) prepared through conventional process.

[0040] DF-1 cells: available from Nansen Central Laboratory of Veterinary Diagnostic techniques Research.

[0041] In-vitro anti-viral effect assay for peptide nucleic acid anti-ALV-J

[0042] The genome of ALV-J was retrieved from the GenBank database, and sequenced by using biological software. By taking the sequence conservation, the percent G+C content, and the base distribution profile into account comprehensively, an antisense nucleic acid was designed by choosing an appropriate region therefrom. The gp85 and P27 genes against the virus finally determined had the following antisense nucleic acid sequences.

TABLE-US-00005 gp85 gp85-1: (SEQIDNO:5) 5-AGACUAAGGCAAAAAUCUGUU-3; gp85-2: (SEQIDNO:6) 5-UAAAUCGGUGUUGUUAUCGCA-3; and gp85-3: (SEQIDNO:7) 5-ACGACUUAUUGAAAAACUCUC-3; P27 P27-1: (SEQIDNO:8) 5-AUAACUCUCAUUAGAUUCGUA-3; P27-2: (SEQIDNO:9) 5-UAUAACCGUCUGUAGUUGGAC-3; and P27-3: (SEQIDNO:10) 5-ACAUAUUUGAUUAUCUCUCCU-3.

[0043] The peptide nucleic acids having the following peptide nucleic acid sequences were artificially synthesized:

TABLE-US-00006 gp85 gp85-1: (SEQIDNO:5) 5-AGACUAAGGCAAAAAUCUGUU-3; gp85-2: (SEQIDNO:6) 5-UAAAUCGGUGUUGUUAUCGCA-3; and gp85-3: (SEQIDNO:7) 5-ACGACUUAUUGAAAAACUCUC-3; P27 P27-1: (SEQIDNO:8) 5-AUAACUCUCAUUAGAUUCGUA-3; P27-2: (SEQIDNO:9) 5-UAUAACCGUCUGUAGUUGGAC-3; and P27-3: (SEQIDNO:10) 5-ACAUAUUUGAUUAUCUCUCCU-3.

[0044] Chitosan-peptide nucleic acid: peptide nucleic acid modified with chitosan through various methods well known in the art, for example, as specifically described in:

[0045] Luessen H L, de leeuw B J, Lang emeyer M, et al. Mucoadhesive polymers in peroral peptide drug delivery. . carbomer and chitosan improve the absorption of the peptide drug buserelin in vivo [J]. Pharm Res, 1996, 13(11): 1668-1172.

[0046] Kotze A F, Luessen H L, de Leeuw B J, et al. Comparison of the effect of different chitosan salts and N-tr-I methyl chitosan chloride on the permeability of intestinal epithelial cells [J]. J Control Release, 1998, 51 (1): 35-46.

[0047] T hanoo B C, Sunny M C, Jayakrishnan A. Crosslinked chitosan microspheres: preparation and evaluation as a matrix for the controlled release of pharmaceuticals [J]. J Pharm Pharmacol, 1992, 44(4): 283-286.

[0048] Portero A, RemunanLo pez C, Criado M T, et al. Reacetylated chitosan microspheres for controlled delivery of antimicrobial agents to the gastric mucosa [J]. J Microencapsul, 2002, 19(6): 797-809.

[0049] The inhibition of the peptide nucleic acid on the target viral gene was detected by using quantitative RT-PCR specific for ALV-J, and the anti-viral titer was determined by viral titer assay.

Day 1:

[0050] Plating: The DF-1 cells, prepared at an earlier stage of digestion, were collected by centrifugation, counted, adjusted to a cell density of 3-610.sup.5 cells/ml with a complete medium (DMEM+5% fetal bovine serum+penicillin), plated in a 24-well plate, and incubated for 18-24 hrs at 37 C. in a carbon dioxide incubator.

Day 2:

[0051] The cell density was microscopically observed. When the cells were grown over to 70-80% of the area of the plate and grown well, the medium was aspirated off, 300 l of the agents (that is, the peptide nucleic acids) to be screened were added per well, each agent having 10 wells. After incubation for 1 hr, 100 l of ALV-J (with the infection rate being 0.01) was added. After 2 hr-adsorption, the unadsorbed viruses were washed off with a nutrient solution, then 4% FBS in DMEM medium was added, and contiuously cultured at 37 C. in 5% CO.sub.2. The cytopathic effect was peridically observed after infection. 72 hrs after infection, the infected cells were repeatedly frozen and thrawed, to release the viruses, and this was used as a sample for virus detection. During experiment, a normal cell control group with no virus and peptide nucleic acid, a positive control group with viruses and no peptide nucleic acid, and a negative control group with peptide nucleic acid and no viruses were also set.

Days 3-5:

[0052] The protection effect of the agent for cells were observed, and the result was evaluated.

Quantitative Detection by Real-Time PCR

[0053] The supernatant of each treatment group was collected, and the viral RNA was extracted by using a total viral RNA extraction kit. The obtained viral RNA was reversely transcripted into cDNA, and then the viral content of the treatment group with ALV-J was detected respectively by using specific Primers. From the results after quantitative amplification, the virus titer and the inhibitory effect of each treatment group in fold differences between the PNA group and the blank control group were calculated by using statistical software.

[0054] In the present invention, ALV-J was quantitatively detected by real-time PCR using primers provided by Huang et al.

TABLE-US-00007 PrimersspecificfordetectionofALV-J Primer1: (SEQIDNO:11) 5-TCAGGACCAAGGGCTTAC-3; and Primer2: (SEQIDNO:12) 5-CTGCCGCTATAACCGTCTG-3. -actinasinternalreference Actin-F: (SEQIDNO:13) 5-TCCCTGTATGCCTCTGGTC-3; and Actin-R: (SEQIDNO:14) 5-TCTCTCTCGGCTGTGGTGG-3.

Reaction System (25 l)

[0055]

TABLE-US-00008 Reagent Amount (l) 2 One-Step SYBR RT-PCR Buffer 12.5 Ex TaqTM HS 0.5 PrimeScriptTM RT Enzyme Mix II 0.5 Forward PCR primer 0.5 Reverse PCR primer 0.5 Total RNA 2 RNase free dH.sub.2O 8.5 In total 25 [0056] Reaction condition [0057] Reverse transcription [0058] 5 min at 42 C. [0059] 10 sec at 95 C. [0060] PCR amplification [0061] Cycle: 40 [0062] 5 sec at 95 C. [0063] 30 sec at 60 C.

[0064] After the reaction was completed, the amplification and melting curves from the Real Time One Step RT-PCR were confirmed, to ensure the specificity and reliability of the results.

In-Vitro Anti-Viral Result of Peptide Nucleic Acid Anti-ALV-J

[0065] The detection results from quantitative PCR show that except for gp85-2 having an unobvious effect, the inhibition rates by gp85-1 and gp85-3 are respectively 78% and 83% (Table 1); and except for P27-1 having an unobvious effect, the inhibition rates by P27-2 and P27-3 are respectively 73% and 77% (Table 1).

TABLE-US-00009 TABLE 1 In-vitro anti-PRRSV effect of peptide nucleic acids for MARC-145 cells Virus inhibition rate Group 72 h Infection and gp85-1 group 78% treatment group gp85-2 group 23% gp85-3 group 83% P27-1 group 17% P27-2 group 73% P27-3 group 77% Virus control group Negative control group Blank control group

[0066] Peptide nucleic acids gp85- and P27- are preferred.

Treatment with Drugs in Combination

[0067] According to the screening result above, the screened drugs having potent anti-viral effect are used in combination on the basis of the experiments above, to compare the difference of the anti-viral effects between the combined agents and a single agent. After the DF-1 cells were infected with ALV-J strain NS-X11, gene drug combinations of gp85 or P27 were added respectively, and a positive, negative, and blank control group were also set. The detection was performed by Real-time PCR, and the virus inhibition rate in each treatment group was statistically analyzed, as described above. The results are shown in Table 2.

TABLE-US-00010 TABLE 2 In-vitro anti-ALV-J effect of various concentrations of peptide nucleic acids for DF-1 cells Virus inhibition rate Group 72 h Infection and gp85-1/3 group 79% treatment group P27-2/3 group 81% gp85-1/3 + P27-2/3 group 88% Virus control group Negative control group Blank control group

Cell Toxicity Test

[0068] 1) The object to be detected was DF-1 cells. 100 l containing 5000 cells was added per well to a 96-well plate. Peptide nucleic acids gp85-1, gp85-3, P27-2 and P27-3 at concentrations of 0.02, 0.1, 0.5, 1, 5, and 10 m were used, each concentration were performed in triplicate. An untreated cell control and a cell free medium control were additionally set.

[0069] 2) After treatment, 10 l of MTT Stock was added per well per 100 l of medium, and continuously incubated for 4 hrs in an incubator at 37 C. Alternatively, the medium was replaced with 100 l of fresh serum-free medium, and then MTT Stock was added.

[0070] 3) The medium was aspirated off, 100 l of MTT lysing agent was added per well, and the volume of the liquid in each well was kept consistent.

[0071] 4) The absorbance (OD) was measured at 570 nm, and comparison and calculation were performed. Note: considering the accuracy, the absorbance (OD) of unreduced MTT was measured at 699 nm, which is then subtracted from OD.sub.570.

[0072] 5) Determination of result: cell proliferation or toxicity=100% (OD.sub.experimentOD.sub.background)/(OD.sub.controlOD.sub.background).

[0073] OD.sub.experiment is the OD value of treated cells, OD.sub.control is the OD value of untreated cells in the control tube, OD.sub.background is the OD value of the cell free medium control. The change in cell proliferation or toxicity after treatment is expressed as percentage of the untreated control.

[0074] The detection result shows that the peptide nucleic acids gp85-1, gp85-3, P27-2 and P27-3 against ALV-J are nontoxic.

Infection and Breeding of Laboratory Animals

[0075] 200 of healthy AA commercial generation broilers aged 1 day (provided by animal farm of Nansen Central Laboratory of Veterinary Diagnostic techniques Research and detected to be ALV-J negative) were assigned to 5 groups at random. The ALV-J infected group (test group) had 40 animals, and the animals were infected by hypodermically inoculating, at the neck, an ALV-J cell culture at a dosage of 0.2 mL/animal at the age of 1 day. The blank control group (control group) had 40 animals, and the animals received no treatment. The animals in the group dosed with a mixture of gp85-+P27- peptide nucleic acids at equal weight ratio (25 ppm, 50 ppm, 100 ppm) were administered via drinking water, and bred with strict isolation. The feedstuff and water for each group were prepared separately without crossing.

[0076] After ALV-J infection, blood was sampled from chickens at various times, and the serum was isolated, and detected for the virus positive rate by ELISA. The result is shown in Table 3.

TABLE-US-00011 TABLE 3 Determination result of ALV-J positive events in serum from animals in each group Time (day) 1 3 5 7 9 Infection and 25 (ppm) + + + treatment group 50 (ppm) + + 100 (ppm) + Infection group without treatment + + + + + Blank control group

[0077] Finally, the preferred dosage is 50-100 ppm.

Growth of Broilers after Viral Infection

[0078] No death occurs in the control group.

[0079] Death occurs in the mixed infection group in 1 week after viral infection.

[0080] The death rates caused by ALV-J infection at week 3 after viral infection are different in each treatment group. The result is shown in Table 4.

[0081] The clinical manifestations, weight gain, immune organ index, and death rate of the broilers were also detected. (1) Clinical manifestations, weight gain, and immune organ index. The disease development and growth of the chicken flocks were observed daily, and the weight of the animals in each group was weighed at days 7, 14, 21, 28, 35, and 49. 5 animals in the virus infected groups and 3 animals in the control group were sacrificed at random weekly, and the thymus, spleen, and bursa of Fabricius was removed and weighed respectively at days 7, 21, 35, and 49. The immune organ index was calculated according to the formula: immune organ index=weight of immune organs (g)/live chicken weight (kg). (2) Statistical calculation of death rate. The number of chickens naturally died was recorded every day, and the death rate in each group was statistically calculated. Moreover, necropsy of the animals was carried out, to observe the lesions. (3) Statistical analysis of data.

TABLE-US-00012 TABLE 4 Influence on weight of broilers at various age of days after ALV-J infection Age Blank in ALV-J infected group control day 25 50 100 group 7 149 32 136 32 129 32 128 23 14 362 49 375 51 392 38 350 38 21 502 55 542 54 532 55 560 54 28 650 75 700 85 670 65 708 65 35 998 89 1088 89 996 92 1260 59 49 1590 120 1688 140 1650 130 2020 45

[0082] As for the influence on the immune organ index of chickens after viral infection, the central immune organ (bursa of Fabricius and thymus) indexes of the ALV-J infected group after 5 weeks are significantly and extremely significantly lower than those of the control group, as shown in 5.

TABLE-US-00013 TABLE 5 Influence on immune organ indexes of broilers after ALV-J infection Group with ALV-J Blank infection group + control Age in day drug treatment group Thymus 2.9 0.32 2.86 0.35 7 Bursa of Fabricius 1.29 0.22 1.65 0.4 Spleen 0.9 19 1.15 0.2 Thymus 3.5 0.36 3.66 0.34 21 Bursa of Fabricius 2.33 0.23 2.65 0.42 Spleen 1.1 0.17 2.55 0.29 Thymus 2.9 0.33 4.86 0.55 35 Bursa of Fabricius 1.33 0.31 1.65 0.4 Spleen 1.5 0.27 1.23 0.32 Thymus 3.1 0.46 5.26 0.17 49 Bursa of Fabricius 0.93 0.18 1.35 0.4 Spleen 1.21 0.23 1.25 0.3