ABSTRACT
Kisspeptin, the neuropeptide produced by Kiss1 neurons in the hypothalamus, is involved in the neuroendocrine regulation of puberty initiation, reproductive system maturation, ovulation and other processes by influencing the secretion of gonadotropin-releasing hormone. Kiss1 gene expression is regulated by multiple trans-regulatory factors and epigenetics. Prediction and preliminary experiments have shown that the seed sequences of miR-92a-3p and miR-25-3p can directly bind to the 3′-UTR of Kiss1 and inhibit the expression of Kiss1. In order to further study the role of miR-92a-3p and miR-25-3p in the regulation of Kiss1, specific absorptive sponge vectors (sponge-miR-92a and sponge-miR-25) with inhibitory effects on miR-92a-3p and miR-25-3p were constructed to realize the functional loss of miRNA. Flow cytometry and dual luciferase reporter assays both confirmed that both sponge vectors could adsorb exogenous or endogenous target miRNAs very effectively. The sponge-miR-92a and sponge-miR-25 vectors are further packaged into the lentivirus LV-sponge-miR-92a and LV-sponge-miR-25. The results of real-time fluorescence quantitative PCR showed that the expression level of Kiss1 in the hypothalamic primary neurons infected by LV-sponge-miR-92a and LV-sponge-miR-25 was significantly up-regulated (P < 0. 05). After injecting LV-sponge-miR-92a into the hypothalamus, the time of female mouse vulva opening was significantly earlier (P<0. 05). The normal oestrus cycle of female mice with was disrupted by injections of LV-sponge-miR-92a and LV-sponge-miR-25 in the hypothalamus. In conclusion, we successfully constructed sponge vectors capable of effectively adsorbing miR-92a-3p and miR-25-3p, and demonstrated their role in removing the inhibition of miR-92a-3p and miR-25-3p on Kiss1. Hypothalamic sponge injection had a certain effect on both the time of vulva opening and the estrus cycle of female mice, suggesting that miR-92a-3p and miR-25-3p may play an important role in the initiation of puberty and reproductive maturity.
ABSTRACT
33 amino acid antibiotic peptide adenoregulin (ADR), which were firstly isolated from the skin of South America arboreal frog Phyllomedusa bicolor, forms alpha-helix amphipathic structure in apolar medium and has a wide spectrum of antimicrobial activity and high potency of lytic ability. Adr gene was cloned in pET32a and transformed into Escherichia coli BL21(DE3) . The cultural and inductive conditions of E. coli BL21(DE3)/pET32a-adr have been optimized. The effect of three factors which were time point of induction, concentration of IPTG in the culture and time of induction on the expression level of Trx-ADR was investigated. The results indicated that the expression level was affected by the time point of induction most predominantly. 9 veriaties of media in which BL21 (DE3)/pET32a-adr was cultured and induced were tested to achieve high expression level of target protein. It was found that glucose in the medium played an important role in keeping stable and high expression level of Trx-ADR. The optimal inductive condition is as follows: the culture medium is 2 x YT + 0.5% glucose, the time point of induction is OD600 = 0.9, the final concentration of IPTG in the culture is 0.1 mmol/L and the induction time is 4 h. BL21 (DE3)/pET32a-adr was cultivated according to the strategy of constant pH at early stage and exponential feeding at later stage to obtain high cell density. During the entire fed-batch phase, by controlling the feeding of glucose, the specific growth rate of the culture was controlled at about 0.15 h(-1), the accumulation of acetic acid was controlled at low level (<2 g/L), but the plasmid stability could not be maintained well. At the end of the cultivation, 40% of the bacteria in the culture lost their plasmids. As a result, the expression level of the target protein declined dramatically, but 90% of Trx-ADR was in soluble form. The expressed fusion protein showed no antibacterial activity, while the native form of ADR lysed from Trx-ADR showed distinct antibacterial activity.