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.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.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.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.