Post Reply - RNase-Resistant Virus-Like Particles

a · J. Mol. Biol.

   FIG. 1. Armored L-RNA packaging system. Two expressing vectors were constructed, in which the maturase and the coat protein were expressed from one plasmid [pET-28(b)] and the pac site and the six-target chimeric RNA sequence were produced from the second plasmid (pACYCDuet-1). The pac site was located between SARS3 and HCV. 3V armored L-RNA was produced by inducing and expressing the two-plasmid system.
FIG. 2. Characterization of the recombinant RNA packaged in armored L-RNA. Recombinant RNA was isolated from 3V armored L-RNA, fractionated in a denaturing 3% agarose gel, stained with ethidium bromide, and detected by using UV fluorescence. Abbreviations: M, RNA marker; 3V, 3V armored L-RNA recombinant RNA.
FIG. 3. Ethidium bromide-stained 1% agarose gel of RT-PCR amplification products of RNA extracted from 3V armored L-RNA and 3V armored RNA. (A) RT-PCR amplification products of RNA extracted from 3V armored L-RNA. Lane 1, negative control with no template; lanes 2 and 3, negative control without RT; lanes 4 and 5, positive control of pACYC-3V plasmid; lanes 6 to 8, RT-PCR of 3V full-length L-RNA. (B) RT-PCR amplification products of RNA extracted from 3V armored RNA: lane 1, positive control of pET-MS2-3V plasmid using the primers S-SARS1 and HA300RT-A; lane 2, RT-PCR of SARS-CoV1 plus SARS-CoV2 plus SARS-CoV3 plus HCV+HA300 using the primers S-SARS1 and HA300RT-A; lane 3, positive control of pET-MS2-3V plasmid using the primers S-SARS1 and HCV-LAP1; lane 4, RT-PCR of SARS-CoV1 plus SARS-CoV2 plus SARS-CoV3 plus HCV using the primers S-SARS1 and HCV-LAP1; lane 5, positive control of pET-MS2-3V plasmid using the primers S-SARS1 and A-SARS3; lane 6, negative control without RT using primers S-SARS1 and A-SARS3; lane 7, RT-PCR of SARS-CoV1 plus SARS-CoV2 plus SARS-CoV3 using the primers S-SARS1 and A-SARS3; lane 8, RT-PCR of SARS-CoV1 plus SARS-CoV2 using the primers S-SARS1 and A-SARS2; lane 9, positive control of pET-MS2-3V plasmid using the primers S-SARS1 and A-SARS2; lane 10, negative control without reverse transcription using the primers S-SARS1 and A-SARS2.
FIG. 4. Stability study of 3V armored L-RNA. 3V armored L-RNA was added to newborn calf serum to a final concentration of 10,000 and 10,000,000 copies/ml. Samples were incubated at 4°C, 37°C, or room temperature for 0, 1, 2, 4, and 8 weeks. Samples were removed at each time point and were stored at ∼80°C until the completion of the experiment. From these materials, we isolated template RNA for real-time RT-PCR assays. Water was used as a negative control. All RNA templates were assayed in a single run by using an HCV RNA PCR fluorescence quantitative diagnostic kit (Shanghai Kehua Bio-Engineering Co., Ltd.). Real-time RT-PCR was conducted by using LightCycler technology (Roche). The mean for low-copy samples was 67,226 IU/ml (4.83 log₁₀; range, 50,100 to 79,400 IU/ml [range, 4.70 to 4.92 log₁₀]), and the coefficient of variation was 12.9%. The mean for high-copy samples was 29,060,000 IU/ml (7.45 log₁₀; range, 22,900,000 to 41,700,000 IU/ml [range, 7.36 to 7.62 log₁₀]), and the coefficient of variation was 22%.
FIG. 5. Calibration of the real-time RT-PCR assay for HCV, SARS-CoV2, and HA300 (H5N1). First, the quantified 3V armored L-RNA was diluted with newborn calf serum 10-fold serially to obtain 100, 1,000, 10,000, 100,000, and 1,000,000 copies/ml. We used the National Reference material for HCV RNA (GBW09151, 2.26 × 10³ IU ml⁻¹ to 4.22 × 10⁷ IU ml⁻¹) to calibrate the serial dilutions of chimeric armored L-RNA and then used the calibrated L-RNA to prepare calibrators for the two real-time RT-PCR assays. From these materials, we isolated RNA template for RT-PCR assays. Newborn calf serum was used as a negative control. Real-time RT-PCR was conducted on a LightCycler thermal cycler (Roche). (A) Log concentration of the international standard for HCV RNA versus the cycle number for the HCV RT-PCR; (B) amplification curve for the HCV RT-PCR assay; (C) amplification curve for the SARS-CoV2 RT-PCR assay; (D) amplification curve for the HA300 (H5N1) RT-PCR assay.
TABLE 1. Primers used for PCR amplification (Table view)Primer name Primer sequence (5′ to 3′)^a
S-MC CGGGATCCTGGCTATCGCTGTAGGTAGCC
A-MC CCCAAGCTTATGGCCGGCGTCTATTAGTAG
S-SARS1 TATCCAAAATGTGACAGAGCCATG
LAP-SARS1 ACGCTGAGGTGTGTAGGTGCAGGTAAGCGTAAAACTCATCCAC
A-SARS2 TAACCAGTCGGTACAGCTACTAAG
LAP-SARS2 AGTTTTACGCTTACCTGCACCTACACACCTCAGCGTTGATATAAAG
A-SARS3 ACTACGTGATGAGGAGCGAGAAGAG
LAP-SARS3 AGCTGTACCGACTGGTTAACAAATTAAAATGTCTGATAATGGACCCC
LAP-SARS2⁺ ATCAGACATTTTAATTTGTTAACCAGTCGGTACAGCTACTAAG
S-HCV ACATGAGGATCACCCATGTGGCGACACTCCACCATAGATCACTC
HCV-LAP1 ATGTAAGACCATTCCGGCTCGCAAGCACCCTATCAGGCAGTAC
A-HA300 GAATCCGTCTTCCATCTTTCCCCCACAGTACCAAAAGATCTTC
HA300LAP CTGATAGGGTGCTTGCGAGCCGGAATGGTCTTACATAGTGGAG
M+SLAP1 ATGGCTCTGTCACATTTTGGATAGAGTAGCTGAGTGCGACCTCCTTAG
M+SLAP2 AAGGAGGTCGCACTCAGCTACTCTATCCAAAATGTGACAGAGCCATG
FIVELAP1 CATGGGTGATCCTCATGTACTACGTGATGAGGAGCGAGAAGAG
FIVELAP2 CGCTCCTCATCACGTAGTACATGAGGATCACCCATGTGGC
OverlapA′ CCTTAATTAA CCCACAGTACCAAAAGATCTTCTTG
M300-S′ TTGGCCGGCC GAGTCTTCTAACCGAGGTCGAAACG
overlap-A CCCACAGTACCAAAAGATCTTCTTG
M300RT-S GGATTTGTATTCACGCTCACC
HA300RT-A TGGGGATGATCTGAATTTTCTC
a
BamHI, HindIII, FseI, and PacI restriction sites are indicated by underscoring; a C vairiant is indicated in boldface type.
TABLE 2. Thermal cycle conditions for the three different kits used in real-time RT-PCR assays (Table view)Program No. of cycles Temp (°C) Incubation time (min:s) Temp transition rate (°C/s) Acquisition mode
HCV          
    1 1 50 25:00 20 None
    2 1 94 2:00 20 None
    3 5 93 3:00 20 None
    55 15:00 2 None
    72 15:00 20 None
    4 42 93 3:00 20 None
    60 45:00 20 Single
    5 1 40 30:00 20 None
H5N1          
    1 1 42 30:00 20 None
    2 1 92 3:00 20 None
    3 5 92 10:00 20 None
    45 30:00 20 None
    72 1:00 20 None
    4 40 92 10:00 20 None
    60 30:00 20 Single
    5 1 40 0:00 20 None
SARS-Cov2          
    1 1 42 30:00 20 None
    2 1 92 3:00 20 None
    3 5 92 10:00 20 None
    52 20:00 2 None
    72 30:00 20 None
  40 92 5:00 20 None
    4   60 30:00 20 Single
    5 1 40 10:00 20 NoneAcknowledgments
This study was supported in part by the SEPSDA project of the European Commission (under no. Sp22-CT-2004-003831), the National Natural Science Foundation of China (30371365 and 30571776), and the Capital Medicine Development Foundation of Beijing (2002-3041).REFERENCES
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