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Topic - RNase-Resistant Virus-Like Particles
Posted: 27 Jan 2022 at 7:51am By reaper
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 namePrimer sequence (5′ to 3′)a
S-MCCGGGATCCTGGCTATCGCTGTAGGTAGCC
A-MCCCCAAGCTTATGGCCGGCGTCTATTAGTAG
S-SARS1TATCCAAAATGTGACAGAGCCATG
LAP-SARS1ACGCTGAGGTGTGTAGGTGCAGGTAAGCGTAAAACTCATCCAC
A-SARS2TAACCAGTCGGTACAGCTACTAAG
LAP-SARS2AGTTTTACGCTTACCTGCACCTACACACCTCAGCGTTGATATAAAG
A-SARS3ACTACGTGATGAGGAGCGAGAAGAG
LAP-SARS3AGCTGTACCGACTGGTTAACAAATTAAAATGTCTGATAATGGACCCC
LAP-SARS2+ATCAGACATTTTAATTTGTTAACCAGTCGGTACAGCTACTAAG
S-HCVACATGAGGATCACCCATGTGGCGACACTCCACCATAGATCACTC
HCV-LAP1ATGTAAGACCATTCCGGCTCGCAAGCACCCTATCAGGCAGTAC
A-HA300GAATCCGTCTTCCATCTTTCCCCCACAGTACCAAAAGATCTTC
HA300LAPCTGATAGGGTGCTTGCGAGCCGGAATGGTCTTACATAGTGGAG
M+SLAP1ATGGCTCTGTCACATTTTGGATAGAGTAGCTGAGTGCGACCTCCTTAG
M+SLAP2AAGGAGGTCGCACTCAGCTACTCTATCCAAAATGTGACAGAGCCATG
FIVELAP1CATGGGTGATCCTCATGTACTACGTGATGAGGAGCGAGAAGAG
FIVELAP2CGCTCCTCATCACGTAGTACATGAGGATCACCCATGTGGC
OverlapA′CCTTAATTAA CCCACAGTACCAAAAGATCTTCTTG
M300-S′TTGGCCGGCC GAGTCTTCTAACCGAGGTCGAAACG
overlap-ACCCACAGTACCAAAAGATCTTCTTG
M300RT-SGGATTTGTATTCACGCTCACC
HA300RT-ATGGGGATGATCTGAATTTTCTC
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)
ProgramNo. of cyclesTemp (°C)Incubation time (min:s)Temp transition rate (°C/s)Acquisition mode
HCV     
    115025:0020None
    21942:0020None
    35933:0020None
  5515:002None
  7215:0020None
    442933:0020None
  6045:0020Single
    514030:0020None
H5N1     
    114230:0020None
    21923:0020None
    359210:0020None
  4530:0020None
  721:0020None
    4409210:0020None
  6030:0020Single
    51400:0020None
SARS-Cov2     
    114230:0020None
    21923:0020None
    359210:0020None
  5220:002None
  7230:0020None
 40925:0020None
    4 6030:0020Single
    514010:0020None

Acknowledgments

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).

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