PEPTIDE NUCLEIC ACID OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND USES THEREOF
20170218370 · 2017-08-03
Inventors
Cpc classification
C12N2320/32
CHEMISTRY; METALLURGY
C12N2770/10022
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present invention discloses a peptide nucleic acid for porcine reproductive and respiratory syndrome virus (PRRSV) and use thereof. The peptide nucleic acid of the present invention is selected from any one or more from the peptide nucleic acids having a) a nucleic acid sequence of Sequence 1 as shown in the Sequencing List; b) a nucleic acid sequence of Sequence 2 as shown in the Sequencing List; c) a nucleic acid sequence of Sequence 3 as shown in the Sequencing List; and d) a nucleic acid sequence of Sequence 3 as shown in the Sequencing List. The peptide nucleic acid of the present invention has no toxic side effect and no resistance, is able to specifically directly inhibit the replication of PRRSV, has a good anti-viral effect, and suffers no food safety problems including drug residue and others.
Claims
1. A peptide nucleic acid, consisting of one or more selected from peptide nucleic acids: a) a nucleic acid sequence of Sequence 1 as shown in the Sequencing List of: TABLE-US-00012 (SEQ ID NO: 1) 5′-AAUAUGAGAGCUGUUGUUGUU-3′; b) a nucleic acid sequence of Sequence 2 as shown in the Sequencing List of: TABLE-US-00013 (SEQ ID NO: 2) 5′-UUAAGUUAUAAAUCAACUGAA-3′; c) a nucleic acid sequence of Sequence 3 as shown in the Sequencing List of: TABLE-US-00014 (SEQ ID NO: 3) 5′-AAUGGAAAACGCCAAAAGCAC-3′; and d) a nucleic acid sequence of Sequence 4 as shown in the Sequencing List of: TABLE-US-00015 (SEQ ID NO: 4) 5′-UCAGAAAGAUCAAAAGGUGCA-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 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 a drug for porcine reproductive and respiratory syndrome virus (PRRSV).
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
[0039] To this end, the following technical solutions are adopted in the present invention.
[0040] PRRSV strain: strain NS-009, available from Nansen Central Laboratory of Veterinary Diagnostic techniques Research.
[0041] Cell line: MARC-145 cells, available from Nansen Central Laboratory of Veterinary Diagnostic techniques Research.
[0042] The genome of PRRSV 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 GP-5 and M gene genes of the virus finally determined had the following antisense nucleic acid sequences.
TABLE-US-00005 GP5: (SEQ ID NO: 6) GP5-1: 5′-AAUAUGAGAGCUGUUGUUGUU-3′ (SEQ ID NO: 7) GP5-2: 5′-UUAAGUUAUAAAUCAACUGAA-3′; and (SEQ ID NO: 8) GP5-3: 5′-AAUGACAAAGCAAAUCAGCGC-3′. M: (SEQ ID NO: 9) M-1: 5′-UGGAAAACGCCAAAAGCACCU-3′. (SEQ ID NO: 10) M-2: 5′-AAUGGAAAACGCCAAAAGCAC-3′.; and (SEQ ID NO: 11) M-3: 5′-UCAGAAAGAUCAAAAGGUGCA-3′.
[0043] The peptide nucleic acids having the following peptide nucleic acid sequences were artificially synthesized:
TABLE-US-00006 GP5: (SEQ ID NO: 6) GP5-1: 5′-AAUAUGAGAGCUGUUGUUGUU-3′ (SEQ ID NO: 7) GP5-2: 5′-UUAAGUUAUAAAUCAACUGAA-3′; and (SEQ ID NO: 8) GP5-3: 5′-AAUGACAAAGCAAAUCAGCGC-3′ M: (SEQ ID NO: 9) M-1: 5′-UGGAAAACGCCAAAAGCACCU-3′ (SEQ ID NO: 10) M-2: 5′-AAUGGAAAACGCCAAAAGCAC-3′; and (SEQ ID NO: 11) M-3: 5′-UCAGAAAGAUCAAAAGGUGCA-3′.
[0044] The inhibition of the peptide nucleic acid on the target viral gene was detected by using quantitative RT-PCR specific for PRRSV, and the anti-viral titer was determined by viral titer assay.
[0045] Day 1:
[0046] Plating: The MARC-145 cells, prepared at an earlier stage of digestion, were collected by centrifugation, counted, adjusted to a cell density of 1×10.sup.5 cells/ml with a complete medium, plated in a 24-well plate, and incubated for 18-24 hrs at 37° C. in a carbon dioxide incubator.
[0047] Day 2:
[0048] 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 μl 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 μl of PRRSV (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. 48 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 virus and no peptide nucleic acid, and a negative control group with peptide nucleic acid and no virus were also set.
[0049] Days 3-5:
[0050] The protection effect of the agent for cells were observed, and the result was evaluated.
[0051] Quantitative detection by Real-time PCR
[0052] 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 PRRSV 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.
[0053] In the present invention, PRRSV was quantitatively detected by real-time PCR using primers provided by Huang et al.
[0054] PRRSV ORF7
TABLE-US-00007 Primer ORF71: (SEQ ID NO: 12) 5′-AAATGGGGCTTCTCCGGGTTTT-3′; and Primer ORF72: (SEQ ID NO: 13) 5′-TCAGCTGTGCCAGATGCTGG-3′. TaqMan probe: (SEQ ID NO: 5) 5′FAM-TCCCGGTCCCTTGCCTCTGGA-TARAM3′.
[0055] β-actin as internal reference
TABLE-US-00008 Actin-F: (SEQ ID NO: 14) 5′-TGACTGACTACCTCATGAAGATCC-3′; and Actin-R: (SEQ ID NO: 15) 5′-TCTCCTTAATGTCACGCACGATT-3′. Actin-Probe: (SEQ ID NO: 16) 5′(FAM)-CGGCTACAGCTTCACCACCACGGC-(TARAM) 3′
[0056] Reaction system (25 μl)
TABLE-US-00009 Reagent Amount (μl) 2 × One-Step RT-PCR Buffer 12.5 Ex TaqT ™ HS 0.5 PrimeScript ™ 0.5 RT Enzyme Mix II Forward PCR primer 0.5 Reverse PCR primer 0.5 Total RNA 2 RNase free dH.sub.2O 8.5 In total 25
[0057] Reaction condition:
[0058] Reverse transcription:
[0059] 5 min at 42° C.
[0060] 10 sec at 95° C.
[0061] PCR amplification:
[0062] Cycles: 40
[0063] 5 sec at 95° C.
[0064] 30 sec at 60° C.
[0065] Viral Titer Detection
[0066] When the cytopathic effect occured, the supernatant of each treated cell culture was collected, serially diluted 10 times to give 7 dilutions, and inoculated in 100 μL/well to MARC-145 cells pre-incubated in a 96-well plate, each sample having 3 replications. The cytopathic effect was observed, until no cytopathic effect was present in the wells. The CCID50PmL (50% cell culture infectious dose) was calculated according to the Karber method.
[0067] The inhibition rate of different peptide nucleic acids on PRRSV replication was calculated according to the formula:
[0068] Assay of Anti-Viral Effect at Various Times
[0069] The infection with viruses and the treatment with drugs were as described above, and the anti-viral effect of GP5-1, GP5-2, GP5-3, M1, M2, and M3 was assayed following the steps above. 24, 36, 48, 60, and 72 hrs after the MARC-145 cells were infected with PRRSV strain NS-009, and 300 μl of peptide nucleic acid to be screened (diluted in complete medium) was added per well, each peptide nucleic acid having 4 parallel wells. The supernatant and cells were collected 24 hrs after treatment with the agent. The copies of PRRSV in each treatment group were quantitatively detected by Real-time PCR using a TaqMan probe, and the virus inhibition rate in the treatment group was statistically analyzed. The result is shown in Table 1.
TABLE-US-00010 TABLE 1 In-vitro anti-PRRSV effect of peptide nucleic acids for MARC-145 cells Virus inhibition rate Group 24 h 36 h 48 h 60 h 72 h Infection and Group GP5-1 55% 67% 76% 77% 82% treatment group Group GP5-2 62% 69% 70% 75% 76% Group GP5-3 25% 39% 40% 42% 43% Group M-1 28% 35% 39% 42% 44% Group M-2 57% 69% 73% 78% 85% Group M-3 52% 66% 72% 76% 80% Virus control group 0 Negative control group 0 Blank control group 0 Peptide nucleic acids GPS-1, GPS-2, M-2 and M-3 are preferred.
[0070] Treatment with Drugs in Combination
[0071] On basis of the experimental results above, the screened drugs having potent anti-viral effect are used in combination, to compare the difference of the anti-viral effects between the combined agents and a single agent. After the MARC-145 cells were infected with PRRSV strain NS-009, gene drug combinations of GP-5 or M were added respectively, and a positive, a negative, and a 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 3.
TABLE-US-00011 TABLE 3 In-vitro anti-PRRSV effect of various concentrations of peptide nucleic acids for MARC-145 cells Virus inhibition rate Group 24 h 36 h 48 h 60 h 72 h Infection and GP5-1 group 60% 55% 68% 62% 60% treatment group GP5-2 group 50% 57% 58% 62% 64% GP5-1 + 2 group 65% 79% 80% 85% 87% M-2 group 47% 49% 53% 58% 55% M-3 group 47% 60% 63% 78% 75% M-2 + 3 group 67% 69% 73% 78% 85% GP5-1 + 2 + 60% 69% 78% 80% 88% M-2 + 3 group Virus control group 0 Negative control group 0 Blank control group 0
[0072] Cell Toxicity Test
[0073] The object to be detected was MARC-145 cells. 100 μl containing 5000 cells was added per well to a 96-well plate. Peptide nucleic acids at concentrations of 0.02, 0.1, 0.5, 1, 5, and 10 μm) were used, each concentration were performed in triplicate. An untreated cell control and a cell free medium control were additionally set.
[0074] After treatment, 10 μl of MTT Stock was added per well per 100 μl of medium, and continuously incubated for 4 hrs in an incubator at 37° C. Alternatively, the medium was replaced with 100 μl of fresh serum-free medium, and then MTT Stock was added.
[0075] The medium was aspirated off, 100 μl of MTT lysing agent was added per well, and the volume of the liquid in each well was kept consistent.
[0076] 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.
[0077] Determination of result: cell proliferation or toxicity=100%×(OD.sub.experiment−OD.sub.background)/(OD.sub.control−OD.sub.background).
[0078] 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.
[0079] The result shows that there is no significant difference (P<0.05%) between the treatment groups with peptide nucleic acids and the control group.
[0080] Animal Test
[0081] PRRSV Strain: Strain NS-009
[0082] Laboratory animals: 100 of healthy pigs aged 25 days were determined by serological and molecular biological examinations to be negative for antigens and antibodies to swine fever virus, Porcine Circovirus, parvovirus, SRRSV, and pseudorabies virus.
[0083] Chitosan-peptide nucleic acid: peptide nucleic acid modified with chitosan through various methods well known in the art, for example, as specifically described in:
[0084] Luessen H L, de leeuw B J, Lang emeyer M, et al. Mucoadhesive polymers in peroral peptide drug delivery. Ö. carbomer and chitosan improve the absorption of the peptide drug buserelin in vivo [J]. Pharm Res, 1996, 13(11): 1 668-1172.
[0085] 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.
[0086] 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.
[0087] Portero A, RemunanLo pez C, Criado M T, et al. Reacetylated chitosan microspheres for controlled delivery of ant-i microbial agents to the gastric mucosa [J]. J Microencapsul, 2002, 19(6): 797-809.
[0088] Grouping:
[0089] Group A: chitosan-peptide nucleic acid (GP5-1+2+M-2+3) added in an amount of 50 ppm;
[0090] Group B: chitosan-peptide nucleic acid (GP5-1+2+M-2+3) added in an amount of 100 ppm;
[0091] Group C: chitosan-peptide nucleic acid (GP5-1+2+M-2+3) added in an amount of 150 ppm; and
[0092] Group D: chitosan-peptide nucleic acid (GP5-1+2+M-2+3) added in an amount of 0 ppm (blank control group).
[0093] Challenge: the animals in groups A, B, C, and D were challenged by intramuscularly injecting 1 ml of viral solution (in which the dosage of the virus was 10.sup.6 TCID50/animal).
[0094] Time at which the chitosan-peptide nucleic acid was added: 6 days before the challenge, the animals were fed on various dosages of chitosan-peptide nucleic acid, and continuously fed for additional two weeks after challenge.
[0095] Development of Disease in Challenged Pigs:
[0096] At day 3 after challenge, pigs in the test groups successively suffered from high fever (body temperature 39-41° C.), followed by symptoms of Porcine Respiratory Disease Complex (PRDC), cyanosis of skin of the ear, stomach, and buttock, general flush, seriously reduced dietary intake of pigs, depression, and failure to stand, and died of failure about 5 days after the occurrence of the symptoms with elapse of time, if no therapeutic measures were taken.
[0097] Test Results:
[0098] 1. Statistical Results of Incidence Rate
[0099] Group A: chitosan-peptide nucleic acid 50 ppm, 6 animals having symptoms of PRDC, incidence rate 24%, and protection rate 76%;
[0100] Group B: chitosan-peptide nucleic acid 100 ppm, 4 animals having symptoms of respiratory syndrome, incidence rate 16%, and protection rate 84%;
[0101] Group C: chitosan-peptide nucleic acid 150 ppm, 3 animals having symptoms of respiratory syndrome, incidence rate 12%, and protection rate 88%; and
[0102] Group D: chitosan-peptide nucleic acid 0 ppm, 23 animals having symptoms of respiratory syndrome, and incidence rate 92%.
[0103] Observation on Lesions of Lymph Nodes by Dissection and Viral Load Test
[0104] 5 days after challenge, compared with the treatment groups with various dosages of chitosan-peptide nucleic acid, the animals in the blank control group have greatly swollen lymph nodes and pulmonary carnification; and the PRRSV load in the serum and lymph nodes of pigs in the treatment groups with various dosages of chitosan-peptide nucleic acid (GP5-1+2+M-2+3) is significantly lower than that in the blank control group.