Preparation of the first batch of national standard of recombinant trypsin / 中国生物制品学杂志

@#Objective To prepare the 1st batch of national standard of recombinant trypsin in order to standardize and improve the quality of recombinant trypsin.Methods Enzyme-substrate identification,HPLC identification,N-terminal sequencing and TOF-MS were used to confirm the property and structure of recombinant trypsin;the purity was determined by HPLC;according to the methods in Chinese Pharmacopoeia(Volume Ⅲ 3603,2020 edition),the specific activity of the candidate standard was determined,and the stability and uniformity were investigated.Results The structure of recombinant trypsin was confirmed,and the specific activity of the 1 st batch of national standard of recombinant trypsin was 5 169 U/mg,containing 80% β-trypsin and 9% α-trypsin.The RSD of purity of α,β-trypsin and retention time of 12 candidate standards were all less than 2.0%.The purity of α,β-trypsin showed no obvious decrease stored at 25 ℃ and relative humidity(RH) 80% for 10 d,while the purity of β-trypsin decreased slightly and the purity of α-trypsin increased slightly stored at 40℃ and RH 80% for 10 d.The purity of β-trypsin decreased slightly when exposed to light(4 000 lx) for 10 d.Conclusion The national standard of recombinant trypsin with accurate structure and high purity was prepared,which can be used for system suitability test of the purity determination.

Development of a robust reporter gene-based assay for the bioactivity determination of recombinant human follicle stimulating hormone (rhFSH) pharmaceutical products.

FSH plays a key role in the function of the reproductive system of human beings and is widely used both diagnostically and therapeutically in reproductive medicine. With the growing incidence of infertility, the demand for FSH pharmaceutical products is increasing. For this reason, the quality control process for FSH products is becoming more stringent. An accurate determination of bioactivity is crucial for the safety and efficacy of recombinant human follicle stimulating hormone (rhFSH). Up to now, in-vivo bioassay based on FSH-induced increases in rat ovarian weight has been the only method widely accepted by different pharmacopoeias. However this method has such drawbacks as the complex procedures, long assay period and high variability. Here, we established a reporter gene assay (RGA) based on the CHO-K1-FSHR-CRE-Luc cell line that stably expresses human follicle stimulating hormone receptor (hFSHR), as well as a luciferase reporter under the control of cyclic adenosine monophosphate (cAMP) response elements (CRES). Our study showed that our new assay not only has good dose-dependent responsiveness to rhFSH, but it also performs excellently in terms of specificity, precision, linearity, and simplicity compared with in-vivo rat bioassays. These results implied that this robust reporter gene assay may be a viable supplement to the animal in-vivo bioassay and may be employed in potency determination of rhFSH pharmaceutical products.

SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5'UTR RNA.

Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5' untranslated region (5'UTR) and a polyadenylated 3'UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3Dpol protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3Dpol, resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5'UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5'UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication.

MicroRNA-26b inhibits hepatitis B virus transcription and replication by targeting the host factor CHORDC1 protein.

Hepatitis B virus (HBV) causes acute and chronic hepatitis in humans, and HBV infection is a major threat to global health. HBV replication is regulated by a series of host factors, including microRNAs (miRNAs), which are highly conserved small noncoding RNAs that participate in a variety of physiological and pathological processes. Here, we report that a chemically synthesized mimic of miR-26b inhibited HBV antigen expression, transcription, and replication, whereas antisense knockdown of endogenous miR-26b enhanced HBV replication in HepG2 cells. Overexpression and knockdown experiments showed that miR-26b significantly decreased HBV enhancer/promoter activities. We identified the cysteine- and histidine-rich domain containing 1 (CHORDC1) as a novel host factor target of miR-26b. CHORDC1 protein but not mRNA was markedly decreased by miR-26b overexpression via base-pairing with complementary sequences in the 3'UTR of its mRNA. Overexpression and knockdown studies showed that CHORDC1 increased viral antigen expression, transcription, and replication by elevating HBV enhancer/promoter activities. Conversely, HBV infection suppressed miR-26b expression and increased CHORDC1 protein levels in human liver cells. Another mature miRNA of the hsa-miR-26 family, miR-26a, had a similar function as miR-26b in targeting CHORDC1 and affecting HBV production. These results suggest that suppression of miR-26b expression up-regulates its target gene CHORDC1, which increases HBV enhancer/promoter activities and promotes viral transcription, gene expression, and replication. Our study could provide new insights into miRNA expression and the persistence of HBV infection.