| Antibacterial peptide(ABP), also called antimicrobial peptide(AMP) or peptide antibiotics, is a group of small molecular peptides easily found in bacteria, virus, various plants and animals. As a kind of polypeptide, controlled by certain genie code and induced under certain outside conditions, ABP can resist the invasion of outside microorganism and kill inner mutant cells. Due to special habitat of amphibian. ABP in its skin plays a very important role in innate defense system. That is the reason why the skin of amphibian is considered as an ideal model for the study of ABP in relationship between its structure and function, inducement and expression.Four kinds of frog, bullfrog {Rana catesbiana }, Rana grylio, Rana temporaria chensinensis and Rana dybowskii Gurnther, were involved in the experiment. Their crude extraction and secretion from skin were isolated respectively through two different ways of inducement and extractionion. The results of their biologic activity detection showed that different secretion from skin of four frogs has different biologic activity. Even for the same secretion from skin of a certain frog, it also differed from each other due to different ways of inducement and extractionion selected. As for their in vitro antibacterial activity, the crude extraction and secretion from skin of four frogs showed a certain antibacterial activity to Bacillus subtilis and most of other experimented bacteria, among which the secretion from skin of Rana catesbiana Shaw was the strongest. No trypsin hydrolytic activity of four frogs was found. Feeble trypsin depressor activity was detected in the crude extraction and the secretion from skin of Rana grylio and bullfrog. All the four crude extraction showed a certain hemolysis activity to red blood cell of rabbit, among which crude extraction from skin of Rana temporaria chensinemis was the strongest. Only the crude extraction from skin of Rana temporaria chensinemis showed feeble hemolysis activity to red blood cell of sheep. Nether the four crude extraction nor the extraction from skin of bullfrog showed bleeding activity. As for the plasm solidification activity detection, all the four crude extraction and the secretion from skin of bullfrog were positive. The crude extraction and secretion from skin of bullfrog, as well as the crude extraction from skin of Rana grylio showed distinct thrombolytic activity. The crude extraction from skin of Rana grylio and bullfrog showed strong (+++) hemoblast anticoagulation activity to blood cell of rabbit, while to blood cell of sheep, they were less strong(++). The crude extraction from skin of Rana dybowskii Gurnther showed much stronger (++++) hemoblast anticoagulation activity to blood cell of sheep. Only the crude extraction from skin of Rana temporaria chensinensis showed hemoblast coagulation activity. None of them four showed distinct hemoblast anticoagulation activity.As for antitumour activity detection at the concentration of 200μg/mL, the crude extraction from skin of bullfrog showed the highly activity one against the growth of Caco-2 cells, while the secretion of bullfrog and the crude extraction of the other three showed feeble toxicity against Caco-2 cells. The crude extraction from skin of bullfrog was the only one against the growth of Hela cells. The crude extraction from skin of Rana grylio showed strong toxicity against MCF-7 cells at the concentration of 200μg/mL, while the other three showed feeble toxicity. Only the crude extraction from skin of bullfrog showed feeble toxicity against SGC-7901 cells, while the other from skin of bullfrog showed feeble toxicity against SGC-7901 cells, while the other three showed no activity. At its safe concentration, the crude extraction from skin of bullfrog, Rana grylio,Rana temporaria chensinensis and Rana dybowskii Gurnther showed different restrainability percentage against SIV as follows in turn, 83.80%, 80.00%, 83.40% and 85.40%. and against LCMV virus, the percentages were 36.60%,21.40%,50.00% and 41.00% respectively.In the process of extractionion and purification of the secretion and crude extraction from skin of bullfrog, 26 apices were collected by Sephadex G-50 gel filtration and RP-HPLC, and 11 out of 26 apices showed antibacterial activity. While for the crude extraction from skin of bullfrog under the same process, 21 apices appeared and 2 out of 21 apices showed antibacterial activity. 3 apices showing antibacterial activity were selected to be analyzed by mass spectrograph.Based on the total RNA isolated from the skin tissue of bullfrog, the cDNA library of skin tissue was constructed with ATripIEx2 as the carrier by using SMART cDNA Library Construction Kit. The obtained original cDNA library contained 1.42*106 pfu/mL , and the percentage of vectors with inserts was 89.5% and the average size of inserts were between 0.5-1.5kb. After amplification, the titer of the library added up to 1.35*109 pfu/ mL.Using technology of RT-PCR, we can clone and gain a nucleotide segment RCABP from the skin of bullfrog, which is 372bp in length. Then, the syncretize expression plasmid were constructed and named pQE-80L/DHFR/RCABP. Positive plasmid was identified and transformed into the competent cell BL21(DE3)which was induced by IPTG. The optimal conditions were determined that the induction time was four hours, the temperature was 37°C, the pH was 7.2 and the IPTG concentration was lmmol/L. Under the conditions, the expressed recombinant protein existed in the form of inclusion bodies, amounting to 31.7% of protein of the induced recombinant bacteria at the presence of IPTG.By detecting the expressed recombinant protein with the method of agar gel diffusion test, the ABP from the skin of bullfrog,expressed in E.coli,showed antibacterial activity in vitro.The rabbit was immunized with the purified expressed recombinant protein as immunogen. The ELISA titer was about 1:6400. After affinity chromatography purification, antibody obtained could specifically bind to the Prokaryotic expressed ABP from the skin of bullfrog by Western blotting analysis. |
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