CATALOGUE Abstract 1 1. Preface 1. 1 SOD Brief introduction 5 1. 2 Human SOD 14 1. 3 The study of cyanobacterium gene engineering19 1. 4 The purperse and meaning of the study 25 2. Material and methods 2. 1 Material 27 2. 2 Methods 32 3. Results and analysis 3. 1 The construction of homologous recombination fragment 42 3. 2 Transform select and identify 47 4. Discussion 4. 1 Whether SOD is suit for oral dosing 59 4. 2 Sited-directed mutagenesis 60 4. 3 The choice of recombine platform 61 4. 4 The detection of the SOD activity 62 4. 5 About the result of mice experiment 64 5. Reference 66 6. Acknowledgement 77 2
Superoxide dismutase,sod (O 2. ), SOD O 2., SOD psod SODhCu,Zn-SOD, Cys 111 Ala 111 groesl rbcs polya Kan r pesodt111 Synechococcus sp.pcc7942 groesl SOD rbcs polya Kan r Synechococcus sp.pcc7942 BG11 SOD DNA PCR 42 SDS-PAGE, SOD HPLC 265nm 3 Western-blot hcu,zn-sod 42 30min SOD 35.34U/ml ; 3.61; 100 g/ml Kan SOD SOD 80 30min 95 SOD 3
MDA hcu,zn-sod 111 Synechococcus sp.pcc7942 DNA hcu,zn-sod hcu,zn-sod hcu,zn-sod hcu,zn-sod 4
Abstracts The main function of superoxide dismutase(sod) is to eliminate the superoxide free ion(o 2. ).So it is widely applied to protect human body from the hurt of oxygen,radiation and postpone consenescence etc. However, the application of Cu,Zn-SOD is limited for its short half life in vivo. The Cys 111 genetic code of hcu,zn-sod gene in the pesod plamid was mutated into the Ala 111 code with sited-directed mutagenesis,and then the plamid pesodt111 which contained groesl promoter, hcu,zn-sod gene, rbcs-polya terminator and reporter gene(kan r ) was constructed. The pesodt111 plamid,in the end, was introduced into cells of Synechococcus sp.pcc7942 with homologous recombination platform, and transformants were screened by Kanamycin. Results of PCR and DNA sequence analysis showed that the target nucleotide had been genetically integrated into genome DNA of the host cell. After induced at 42, the hcu,zn-sod protein was detected by SDS-PAGE HPLC and Western Blot in the transgenic algae. The Pyrogallol autoxidation assay revealed that its SOD activity was 35.34 U/mL culture medium. And the result of protein scan shows the aimed protein is 3.61of the total dissoluble 5
protein. In addition, the transgenic algae keep alive in 100 mg/ml Kan and the expressed mutated hcu,zn-sod protein can stand higher temperature. After oral administration of transgenic algae for 10 days, the mouse serum total antioxidant ability was increase remarkably,and its MDA content was reduced significantly. In this study the hcu,zn-sod gene was sited-directed mutated and had successfully expressed in the Synechococcus sp.pcc7942.the expressing product has the activity of native hcu,zn-sod and stability.the animal experiment also showed the transgene algae could enhance animal s antioxidative ability.the study provide a basis for further research of oral hcu,zn-sod with longer half life. Key words: hcu,zn-sod, sited-directed mutagenesis, Synechococcus sp.pcc7942, expression 6
1 SOD 1938 Mann Keilin ( Hemocuprein) JoeM.Mccord [1] 1969 (EC.15.1.11,Superoxide dismutase, SOD) O 2.,,,, 1.1 SOD SOD SOD Cu,Zn-SODMn-SOD Fe-SOD Mn-SOD Fe-SOD Co SOD Ni SOD [2] Mn-SOD Fe-SOD SOD Mn-SOD Mn-SOD 94% 43% SOD SOD, SOD Cu,Zn-SOD SOD 7
1.2 1.2.1, Cu,Zn-SOD, ESRHNMR X Cu,Zn-SOD Cu,Zn-SOD 32KD, 2Cu 2 Zn Cu,Zn-SOD,, 1Cu1 Zn 1 [3]. 1982,Tainer [4] Cu,Zn-SOD0.2 nm,, (-barrel) (loop) Cu,Zn-SOD, 444661118, Zn616978 81 CuZn 61, CuZn, Cu 0.03nm, 81, 61, CuZn, 122 69 44, CuZn 8
55144SH Cu,Zn-SOD, Cu,Zn-SOD 1. Cu,Zn-SOD Fig.1 Illustration of the Bovine erythrocyte copper and zinc location 1.2.2 X, Cu 2+ 4 His 1 H 2 O Zn 2 3 His 1 Asp Cu 2+ Zn 2+ 1 His 15 9 6 Cu Zn O 2 His Cu 2+ 6 Arg143 9
O 2- H + O 2-99 Cu 2+ Zn 2+ Cu,Zn-SOD, Cu 2+, 44 46 61 118 His,, Cu 2+ Cu 2+ 2+,Cu 2+ Zn 2+ 1 His 61, Cu 2+, Zn 2+, Zn 2+ Co 2+ Hg 2+ Cd 2+ [5] 1.2.3 Cu,Zn-SOD, 32kD, 2 1 Zn Cu ph Cu,Zn-SOD 75,, 95, Cu,Zn-SOD, (Tm) Cu,Zn-SOD Cu,Zn-SOD ph5.3-10.5 [6] 1 Cu,Zn-SODMn-SOD Fe-SOD 1.SOD Table 1.The character of SOD 10
SOD Cu,Zn-SOD Mn-SOD Fe-SOD 32000 1Cu:1Zn, 65000 42000 0.5~1Mn, 85000 42000 0.5~1Fe, 85000 1.2.4 Cu,Zn-SOD,, ph=5.29.6 ph,,, : Cu(II) Cu(I), O 2, His-61 Cu ; 11 (nm) 260 680 280 475 CN - H2O2+E DTA R(N3)X 280 R(N3)X H2O2+E DTA
Cu(I) Cu(II),, His-61, His-61 [7] SOD 2-., SOD,SOD X-, SOD 1.2.5 Cu,Zn-SOD NCBI Cu,Zn-SOD mrna Cu,Zn-SOD Cu,Zn-SOD Pantroglodytes Cu,Zn-SOD Cu,Zn-SOD 15 Macacafascicularis Cu,Zn-SOD 22 8 Cu,Zn-SOD 85 Cu,Zn-SOD 12
2 Cu,Zn-SOD mrna Cu,Zn-SOD mrna Fig.2 The compare map between Canis familiaris Cu,Zn-SOD mrna and Homo sapiens A: Canis familiaris Cu,Zn-SOD mrna B: Homo sapiens Cu,Zn-SOD mrna 13
Cu,Zn-SOD 3 Cu,Zn-SOD mrna Cu,Zn-SOD mrna A: Bovine Cu,Zn-SOD mrna B: Homo sapiens Cu,Zn-SOD mrna 14
383 Cu,Zn-SOD 82 Cu,Zn-SOD 1.3 SOD 1SOD Zweier [8], O 2-. O 2-., Ca 2+ O 2-., SOD [9] SOD [10] SOD,,,,,, SOD SOD BTN Cu,Zn-SOD DDIP Cu,Zn-SOD - E.coli Cu,Zn-SOD PEG SOD 15
10 Chiron SOD [11] 2 SOD SOD SOD SOD SOD SOD [12] 3SOD SOD SOD, SODSOD SOD [13] SOD SOD SOD 2.SOD 2.1 SOD 3 SOD Cu,Zn-SODMn-SOD EC-SOD( 3 SOD SOD1,SOD2,SOD3) Cu,Zn-SOD 2 Mn-SOD 4 EC-SOD Marklund S L 1982 Cu,Zn-SOD, Cu,Zn-SOD 4 [11415] 2.2 SOD 2.2.1 SOD 16
SOD, SOD,, SOD SOD Hallewell [16] 1985 hcu,zn-sodcdna [17] Cu,Zn-SOD 1987hMn-SODcDNA hec-sod [18] SOD Hartman [19] 1986 hcu,zn-sod, 10 N- Yasunobu [20] rbc PBAX18R PCC6301 hcu,zn-sod 3 L NS20Y hcu,zn-sod [21] hcu,zn-sod hcu,zn-sod [22], 1 g 40 µg hcu,zn-sod 0.62 hcu,zn-sod hmn-sod EC-SOD [23][24] hcu,zn-sod [25][26] (RT-PCR), Cu,Zn-SODcDNA hmn-sodcdnaec-sodcdna hcu,zn-sodcdna T7 Pet-22b, petcu,zn-sod, BL21(DE3) 1mmol/L 17
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