RESUMO
@#ObjectiveTo investigate the effect of cryopreservation on the biological activity of the national standard of human granulocyte colony-stimulating factor(hG-CSF)after reconstitution,so as to provide a reference for the use of the national standard of hG-CSF.MethodsThe biological activity of the standard was determined according to the general rule 3525 of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition);The reconstituted hG-CSF national standard was aliquoted and stored at-80 ℃,-40 ℃ and-20 ℃,and then sampled at 1,2,3,5 and 6 months to detect the biological activity. The standards reconstituted before use were used to quantify the standards stored at -80 ℃ for different time durations,and the standards stored at -80 ℃ were defined as the reference of 100% activity to quantify the relative biological activity of the other samples.ResultsThe Eyrlng equation fitted by the thermal acceleration stability experiment was:ln {k(t)} = 6. 75-3 772. 20/T + ln(T),R~2= 0. 969. The biological activity of hG-CSF national standard was predicted to decrease by 5% after about 93. 4 months storage at -80 ℃；The biological activity of reconstituted standards frozen at -80 ℃ decreased by about 24%.ConclusionThe aliquoted reconstituted hG-CSF national standard can be stored at -80 ℃ stably for more than half a year. However,freezing and thawing will cause the activity value to drop by more than 20%,so it is not recommended to reuse after reconstitution.
RESUMO
Owing to the outbreak of the novel coronavirus (SARS-CoV-2) worldwide at the end of 2019, the development of a SARS-CoV-2 vaccine became an urgent need. In this study, we developed a type 9 adeno-associated virus vectored vaccine candidate expressing a dimeric receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) and evaluated its immunogenicity in a murine model. The vaccine candidate, named AAV9-RBD virus, was constructed by inserting a signal peptide to the N-terminus of two copies of RBD, spaced by a linker, into the genome of a type 9 adeno-associated virus. In vitro assays showed that HeLa cells infected by the recombinant AAV virus expressed high levels of the recombinant RBD protein, mostly found in the cell culture supernatant. The recombinant AAV9-RBD virus was cultured and purified. The genome titer of the purified recombinant AAV9-RBD virus was determined to be 2.4 × 1013 genome copies/mL (GC/mL) by Q-PCR. Balb/c mice were immunized with the virus by intramuscular injection or nasal drip administration. Eight weeks after immunization, neutralizing antibodies against the new coronavirus pseudovirus were detected in the sera of all mice; the mean neutralizing antibody EC50 values were 517.7 ± 292.1 (n=10) and 682.8 ± 454.0 (n=10) in the intramuscular injection group and nasal drip group, respectively. The results of this study showed that the recombinant AAV9-RBD virus may be used for the development of a SARS-CoV-2 vaccine.