568 42, cdna,, T3 T7 P1(5 -CTGTTCCGTCAGGTAGTGGG-3 ) P2(5 -CTGCTGGATGCTTTCAAGGT-3 ) 5 3 : 94 5min 94 30s 55 30s 72 30s, 30, 72 10min 1% (W/V)

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1 42 4 Vol.42, No OCEANOLOGIA ET LIMNOLOGIA SINICA July, 2011 南移养殖仿刺参 (Stichopus japonicus Selenka) * 铁蛋白基因的克隆及表达特征分析 , 2 ( ; ) cdna RACE PCR Western blot,,, cdna 1222bp, 187bp 5 UTR; 513bp 3 UTR bp ;, 2, Western blot,,,, PCR, Western blot Q346 (ferritin), (, 2010), (Wang et al, 2009) (Li et al, 2008b) (Qiu et al, 2008) (Huang et al, 1996) (von Darl et al, 1994) (Zhang et al, 2003) (, 2009) (, 2008) H (Stichopus japonicus Selenka),, cdna RACE, cdna, PCR, 1 1.1, 4 BALB/c RNA cdna pmd18-t Taq DNA Polymerase DNA Marker TaKaRa, SYBR Premix Ex Taq TM II E. coli BL21 pet-28a(+) IgG, Sigma, *, 2007GB2C ; ( ), 2008C ;, 2007C30001 ;, 2011A ;, xk111087,, lichenghua@nbu.edu.cn :,,, litaiwu@nbu.edu.cn : , :

al, 2008) (Huang et al, 1996) (von Darl et al, 1994) (Zhang et al, 2003) (, 2009) (, 2008) H (Stichopus japonicus Selenka),, cdna RACE, cdna, PCR, 1 1.

2 568 42, cdna,, T3 T7 P1(5 -CTGTTCCGTCAGGTAGTGGG-3 ) P2(5 -CTGCTGGATGCTTTCAAGGT-3 ) 5 3 : 94 5min 94 30s 55 30s 72 30s, 30, 72 10min 1% (W/V), pmd18-t,,, BLAST( ( cdna SMART ( SWISS-MODEL ( expasy.org/) mrna PCR P3 (5 -CGAGTCGTGGGAGATTTTT-3 ) P4 (5 -CACTT TGGCTGCTTTGAAC-3 ) 18S rrna, P5 (5 -CTGCTGGATGCTTTCAAGGT-3 ) P6 (5 -CTGT TCCGTCAGGTAGTGGG-3 ), PCR : 94 5min 94 15s 56 15s 72 20s 40, 72 10min PCR,, Rotor-Gene CT, CT P7 (5 -GGA TCCATGCAGCCAAGCCAAGTC-3 ) P8 (5 -AAGC TTTTAATCCTCCTTC AGGTT-3 ) ( BamH Hind ), cdna, PCR : 94 5min, 94 30s, 58 30s, 72 1min, 35, 72 10min BamH Hind pet-28a(+),, E. coli BL21, LB ( 50 g/ml ), 37 OD mmol/L IPTG, 37, h 1mL 12000r/min 5min, 40 l ph 7.4 PBS, 10 l 5, 10min,, SDS-PAGE mol/L KCl SDS-PAGE,, PBS, 5 BALB/c 100 g/, 75 g/, 50 g/, 75 g/,, 37 30min, r/min 10min,, Western blot 4h E. coli BL21,, 1 5 PBS, r/min 15min, 5, 10min, SDS-PAGE, PAGE 20V, 4 PVDF 5% ( TBST ) 4h, E. coli BL21 4 8h, 2h, TBST 3, 10min, TBS 10min; 1h, TBST TBS, NBT/BCIP cdna 5 RACE, cdna, P1 T3, 604bp, 3 RACE, P2 T7

3 mrna PCR P3 (5 -CGAGTCGTGGGAGATTTTT-3 ) P4 (5 -CACTT TGGCTGCTTTGAAC-3 ) 18S rrna, P5 (5 -CTGCTGGATGCTTTCAAGGT-3 ) P6 (5 -CTGT TCCGTCAGGTAGTGGG-3 ), PCR : 94 5min 94 15s 56 15s 72 20s 40, 72 10min

3 4 : (Stichopus japonicus Selenka) 569, 804bp EST, 1222bp cdna cdna 1222bp, 187bp 5 (5 UTR), 513bp 3 UTR, 522bp (ORF) 5 UTR, CAGTGA, 6 C (Jin et al, 2011) 3 UTR 25bp Poly(A) AATAAA( 1) ORF 173, ExPASy Proteomics tools 20.0kDa 5.42; EEREHA EKLMKYQN KRGGR, 7 (Glu-25, Tyr-32, Glu-59, Glu-60, His-63, Glu-105 Gln-139), 4 (His-55, Glu-57, Glu-58 Glu-61), 3 (His-116, Tyr-129 Glu-131), NQSL( 1) H,, ( 2) 2.3 BLAST, Holothuria glaberrima (ABS29643) Asterias forbesii (AAB60883) Haliotis discus hannai (ABY87353) Crassostrea gigas (CAD91440) Argopecten irradians (ADR 71732), Danio rerio (NP ),, 3 UTR (, 2010), 79%,, 2.4 MEGA 4.1,, 1 cdna Fig.1 Full-length ferritin cdna of S. japonicus (SjFER) and its deduced amino acid sequence : ; N- ; ; * 2 H Fig.2 Structural prediction of ferritin from S. japonicus and human using Swiss-Model A. ; B. H

42; EEREHA EKLMKYQN KRGGR, 7 (Glu-25, Tyr-32, Glu-59, Glu-60, His-63, Glu-105 Gln-139), 4 (His-55, Glu-57, Glu-58 Glu-61), 3 (His-116, Tyr-129 Glu-131), NQSL( 1) H,, ( 2) 2.

4 (neighbour-joining method), 3 :, ;, 24kDa,,,,,, 4 mrna Fig.4 Tissues expression profiles analysis of ferritin from North and South cultured S. japonicus 3 NJ Fig.3 Unrooted phylogenetic tree of ferritins based on NJ method 2.5, 18S rrna ( 4),, (P<0.01), SDS-PAGE, 5A IPTG, 5 SDS-PAGE (A) Western blot (B) Fig.5 SDS-PAGE analysis of recombinant product of S. japonicus ferritin (A) and specificity of ferritin polyclonal antibody determined by western blot (B) A: 1. ; 2 4. ; h 2h 3h 4h B: 1. ; 2. ; 3. ; 4. ; Western blot 2.6,, Western blot, 5B 24kDa E. coli BL21,, PBS,,,

6 SDS-PAGE, 5A IPTG, 5 SDS-PAGE (A) Western blot (B) Fig.5 SDS-PAGE analysis of recombinant product of S.

5 4 : (Stichopus japonicus Selenka) 571, 3,, BLAST, H, NJ H,,,,,,, (Masuda et al, 2007),, Clemens (1989), 12, ;,, Chen (2003) Northern blot (Artemia franciscana),,, 16h, 3 UTR ( 137bp, 513bp), 3 UTR (Li et al, 2008a) PCR mrna RNA, PCR (, 2007; Neuvians et al, 2005), -actin 28S rrna 18S rrna GAPDH TBP beta-2-microglobulin (Bustin, 2000), 18S rrna ( ), ;, RNA 80%, mrna, (Blanquicett et al, 2002) 18S rrna Real-time PCR,,, 2,, 25,,,,, Western blot,,, Western blot,,,,, actin PCR., 34(2): 73 75,,, (Neobenedenia melleni) cdna., 41(1): ,,, (Phascolosoma esculenta)., 39(3): ,,, , 34(6): ,,, (Tegillarca granosa) cdna (Ferritin)., 40(3): Blanquicett C, Johnson M R, Heslin M et al, Housekeeping gene variability in normal and carcinomatous colorectal and liver tissues: applications in pharmacogenomic gene expres-

18S rrna Real-time PCR,,, 2,, 25,,,,, Western blot,,, Western blot,,,,, 2007. -actin PCR., 34(2): 73 75,,, 2010. (Neobenedenia melleni) cdna., 41(1): 121 125,,, 2008. (Phascolosoma esculenta).

6 sion studies. Anal Biochem, 303(2): Bustin S A, Absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 25: Chen T, Amons R, Clegg J S et al, Molecular characterization of artemin and ferritin from Artemia franciscana. Eur J Biochem, 270(1): Clemens L E, Basch P F, Schistosoma mansoni: effect of transferrin and growth factors on development of schistosomula in vitro. J Parasitol, 75(3): Huang T, Law J H, Söderhäll K, Purification and cdna cloning of ferritin from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus. Eur Biochem, 236: Jin C, Li C, Su X et al, Identification and characterization of a Tegillarca granosa ferritin regulated by iron ion exposure and thermal stress. Dev Comp Immunol, 35: Li C, Ni D, Song L et al, 2008a. Molecular cloning and characterization of a catalase from Zhikong scallop Chlamys farreri. Fish Shellfish Immunol, 24 (1): Li M, Saren G W, Zheng S, 2008b. Identification and expression of a ferritin homolog in amphioxus Branchiostoma belcheri: Evidence for its dual role in immune response and iron metabolism. Com Biochem Physiol B, 150: Masuda T, Goto F, Yoshihara T et al, Construction of homo- and heteropolymers of plant ferritin subunits using an in vitro protein expression system. Protein Expr Purif, 56: Neuvians T P, Gashaw I, Sauer C G et al, Standardization strategy for quantitative PCR in human seminoma and normal testis. J Biotechnol, 117(2): Qiu G, Zheng L, Liu P, Transcriptional regulation of ferritin mrna levels by iron in the freshwater giant prawn, Macrobrachium rosenbergii. Com Biochem Physiol B, 150: von Darl M, Harrision P M, Bottke W, cdna cloning and deduced amino acid sequence of two ferritins: soma ferritin and yolk ferritin, from the snail Lymnaea stagnalis. Eur J Biochem, 222: Wang D, Kim B Y, Lee K S, Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignites. Com Biochem Physiol B, 152: Zhang Y, Meng Q, Jiang T et al, A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata). Com Biochem Physiol B, 135: CLONING AND CHARACTERIZATION OF FERRITIN GENE FROM SOUTH CULTURED STICHOPUS JAPONICUS LI Cheng-Hua 1, CUI Jing 1, LI Ye 1, ZHOU Jun 1, SU Xiu-Rong 1, LI Tai-Wu 1, 2 (1. Faculty of Life Science and Biotechnology, Ningbo University, Ningbo, ; 2. Ningbo City College of Vocational Technology, Ningbo, ) Abstract With the approaches of cdna library, RACE, fluorescent real-time quantitative PCR and western blot, the full-length ferritin cdna was identified and characterized in South cultured Stichopus japonicus (denoted as SjFER) in the present study. All these results indicated: 1) The cdna of SjFER was of 1222bp, consisting of a 5'UTR of 513bp with a putative iron regulatory element (IRE), a 3'UTR of 187bp, and a complete open reading frame of 522bp encoding a polypeptide with 173 amino acid residues. The conserved motifs for ferritin including iron binding signature, H-specific ferroxidase center and phosphorylation site were totally found in the deduced amino acid of SjFER. 2) SjFER was an important molecule in the species environmental adaption. The expression level of SjFER in South sea cucumber was two times higher than that from North in the tissue of muscle, while no difference occurred in tissue of respiratory tree. 3) The ployclonal antibodies generated from the recombinant product of SjFER could be specifically identified not only the recombinant product, but also the native protein from muscles, and should be used for next functional validation. Key words Stichopus japonicus, Ferritin, Fluorescent real-time quantitative PCR, Western blot

Eur J Biochem, 270(1): 137 145 Clemens L E, Basch P F, 1989. Schistosoma mansoni: effect of transferrin and growth factors on development of schistosomula in vitro.

2004级研究生现代免疫学实验技术结业报告

2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)