| 其他摘要 | We have separated one neurotoxin, named as Anntoxin, and one stem cell self-renewal promoting factor, named as AnSF, from the skin secretions of Hyla annectans annectans (Jerdon) firstly. And then we get the encoding cDNA sequences of Anntoxin and AnSF in the constructed cDNA library through designing special primers of Anntoxin and AnSF. The Anntoxin cDNA sequence’s Genebank number is FJ598043, and the AnSF cDNA sequence is not defined now. Anntoxin has 60 amino acid residues, and has a classical Kunitz solution structure through Tridimensional Nuclear Magnetic Resonance (3D-NMR) test. But Anntoxin has only two (motif: 1-4, 2-3) out of the three (motif: 1-6, 2-4, 3-5) disulfide bridges in the normal Kunitz protease inhibitors. AnSF has 123 amino acid residues, and it can promote human embryonic stem cells (hESCs) and rhesuss monkey nervous stem cells (rNSCs) self-renewal. Getting enough protein to test its biology functions in vivo and in vitro and analyze its solution structure through 3D-NMR, we expressed Anntoxin in vitro successfully. The rAnntoxin has nearly similar bio-activities to native Anntoxin separated from the skin secretions of H. annectans. It can inhibit the hydrolysis activity of tripsin to its substrate strongly too. Through 3D-NMR the solution structure of Anntoxin is constructed, which proves Anntoxin has classical Kunitz structure though it only possesses two out of three disulfide bonds as normal Kunitz serine protease inhibitors, and the NMR number is PDB ID 2KCR and BMRB ID 16094. Anntoxin has similar sequence of 32.8% and 36.7% with Conkunitzin-S1, originating from Cone Snail, and δ-DaTX, separated from the venom of black cobra (Dendroaspis polylepis polylepis), respectively. It also has some homology to Stonustoxin from fish. Using patch-clamp technology, we test the effect of Anntoxin on voltage-gated ion channels such as K+ channel, Na+ channel, and Ca2+ channel on rat dorsal root ganglion (DRG). It is proved that Anntoxin has strong inhibition effect on the tetrodotoxin sensitive (TTX-S) Na+ channel, and little effect on K+ channel and Ca2+ channel. Then we test the effect of Anntoxin on sub-type K+ channels such as Kv1.1, Kv1.2, Kv1.3, Kv2.1, Kv4.2 and Kv4.3, expressed on Xenopus laevis oocytes, and we get negative result. According to the result of RT-PCR, WesternBlot, and enzyme-linked immunosorbent assay (ELISA), though mRNA of Anntoxin can be detected in skin, brain, stomach, liver and intestine (RT-PCR), there are 29.5, 5.39, 4.80, and 2.04 μg/g wet fresh tissues (WT) expressed in the skin, brain, stomach, and liver, respectively (ELISA). This shows that Anntoxin is expressed abundant in skin, ten folds more than in other tissues. Because skin is the most important interface between the toad internal milieu and the outer environment, and acts as the first barrier against microorganism, blood-sucking insects, birds, snakes, and other mammals, highly expressed Anntoxin in the skin may furnish skin with a strong chemical weapon to fulfill its functions. As the result of acute toxicity of Anntoxin to the third instar worm of Laphygma exigua Hubner, Enhydris plumbea, Coturnix coturnix, and Kunming Mice, the LD50 is 50, 450, 2 500, and 3 000 μg/kg bodyweight, respectively. It shows that Anntoxin can kill these potential natural enemies in H. annectans’ environment. Through the same method as obtaining rAnntoxin, we get recombinated AnSF (rAnSF). After released from the fusion His-tagged protein by formic acid, the rAnSF is centrifuged in Amicon Ultra-4 Centrifugal device (10K, 30K), and then the retenate solution is uploaded in C8 (30×0.46 cm), and through RP-HPLC we get highly pure AnSF. AnSF is cocultured with human embryonic stem cells (hESCs) and rhesus monkey nervous stem cells (mNSCs), respectively, at three concentrations of, i.e. 10, 100, and 500 ng/ml. We found that between the 10 and 100 ng/ml AnSF can sustain hESCs self-renew, so do 10 ng/ml AnSF to mNSCs. High concentration (500 ng/ml) of AnSF has heavy cyto-toxicity to both hESCs and mNSCs. It is investigated from the RT-PCR result, tested in skin, muscle, liver, pancreas, stomach, intestine, heart, and brain, only skin expresses a little AnSF. This shows AnSF may act as key factor to sustain the skin stem cell pool, keeps homeostasis in the skin, and provides enough skin stem cell to differentiate into mature skin cells, which need to be changed quickly, because skin should secrete many peptides and proteins involved in the innate immunity system and other physiological processes. |
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