[Construction and expression of virus-like particles containing rotavirus NSP3 gene].
OBJECTIVE
To construct and express ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene  by changing the affinity of MS2 bacteriophage coat protein pac site and to discuss the stability.


METHODS
In the study, 1 049 bp  rotavirus NSP3 gene fragments were amplified by PCR using the primers containing PvuI and KpnI restriction enzyme sites and  the uridine at position -5 in the pac site was replaced with cytosine to increase the affinity. The gel-purified PCR-amplified DNA  fragments and pACYC-MS2 vector were digested with PvuI and KpnI and then ligated to generate recombinant plasmid  pACYC-MS2-NSP3. The expression vector was transformed into competent Escherichia coli strain TOP 10, and was verified by  PCR and sequencing. The positive bacteria were transformed into competent E.coli strain BL21(DE3). Then the cells were lysed by  ultrasonic disruption, virus like particles (VLPs) were harvested after purification and their stability was discussed.


RESULTS
The  expression plasmid containing mutant pac site (uridine at position -5 in the pac site replaced with cytosine) was constructed  successfully and the ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene were expressed successfully. The  VLPs were resistant to ribonuclease and deoxyribonuclease, and were stable at -20 °C, 4 °C and room temperature(25 °C),  respectively.


CONCLUSIONS
The methods used to increase the affinity of pac site could successfully construct and express the  VLPs. The VLPs containing rotavirus NSP3 gene are stable and could be used as surrogates for positive controls and standards in  rotavirus real time fluorescent quantitative reverse transcription-PCR kits.
Graduate School of Peking Union Medical College, Beijing, China.
Le
L
Chang
Kuo
K
Zhang
Rui
R
Zhang
Jie-hong
JH
Xie
Jin-ming
JM
Li
Lu-nan
LN
Wang
D017909:Levivirus / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction; D012260:Ribonucleases / Q000235:genetics; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014771:Virion / Q000235:genetics*
C082740:NSP3 protein, Rotavirus; D017361:Viral Nonstructural Proteins; D012260:Ribonucleases