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1 刺激隐核虫甘油醛 -3- 磷酸脱氢酶的分子特征 1, 付国良 2, 单金红 1, 胡燕红 1, 谢金珠 1 1, 2 1, 晏燕花, 黄晓红 (1.,, ; 2., ) 摘要 : 从刺激隐核虫 (Cryptocaryon irritans) 滋养体 / 包囊前体 cdna 文库中筛选出了甘油醛 -3- 磷酸脱氢酶基因 (CiGAPDH), 定点诱变 CiGAPDH 基因开放阅读框内的非通用密码子后, 构建其原核表达载体 pgex-4t-3/cigapdh, 转化到大肠杆菌 BL21(DE3) 中, 用异丙基硫式 -B-D- 半乳糖苷诱导表达, 结果大肠杆菌成功表达了 rcigapdh 蛋白用抗 rcigapdh 蛋白的鼠血清进行免疫印迹分析, 结果抗血清能够识别刺激隐核虫各期虫体的天然 CiGAPDH 蛋白, 其表观分子质量为 37.3 ku, 与根据氨基酸序列推算的理论值相符 ; 实验表明 rcigapdh 蛋白具有很好的免疫原性, 而且 CiGAPDH 在生活史的各阶段均有表达, 符合持家基因的特征利用间接免疫荧光抗体实验 (IFAT) 检测天然 CiGAPDH 蛋白在刺激隐核虫幼虫上的定位, 结果表明天然 CiGAPDH 蛋白主要分布在幼虫的细胞质内, 且在胞口位置分布最多对 CiGAPDH 的进一步研究可能为刺激隐核虫感染的药物靶点的寻找多条线索关键词 : 刺激隐核虫 (Cryptocaryon irritans); 甘油醛 -3- 磷酸脱氢酶 ; 原核表达 ; 定位中图分类号 : 文献标识码 : A 文章编号 : (2016) doi: /hykx (Cryptocaryon irritans), (Prostomatea) (Prorodontida) (Crytocaryonidae) (Cryptocaryon) [1] 1937 Sikama [2], (trophonts) (protomonts) (tomonts) (theronts) 4 [3],, [4], (Siganus oramin), [5-6], [3, 7] [3], ( ),, 200,,,,,, [8] [9],,,,,,,, -3- (glyceraldehyde-3-phosphate dehydrogenase, GAPDH),, ATP [10], [11] GAPDH, RNA (Western blot) [12] ; 3-1, 3- : ; : : JK (JK ); (2014J01120); (2013NZ0002-5) [Foundation : The work was supported by grants from JK project from Fujian Provincial Department of Education (JK ), Natural Science Foundation of Fujian Province (2014J01120), and Major Project in Agriculture of Fujian Province (2013NZ0002-5)] : (1988-),,,,, fu-guo-liang@163.com;,,, biohxh@fjnu.edu.cn Marine Sciences / Vol. 40, No. 2 /

2 , GAPDH, GAPDH,, DNA DNA [13-14] [15] [16],, GAPDH, GAPDH, -3- (CiGAPDH) cdna EST, GAPDH (Genbank : KP662711), pgex-4t-3/cigapdh, E. coli BL21(DE3),, CiGAPDH, 1 材料与方法 1.1 材料 XP , 鮋 (Sebastiscus marmoratus),, (specific pathogen free, SPF) XP7.2 cdna ptripiex2/cigapdh Escherichia coli BM25.8 E. coli DH5α E. coli BL21(DE3) pgex-4t CiGAPDH 基因序列及其蛋白的分子进化分析 EST, ptripiex2 ( 5 - CTCCGAGATCTGGACGAGC-3, 5 -TAA TACGACTCACTATAGGG-3 ) Genbank, GAPDH, CiGAPDH,, Mega4.0 NJ CiGAPDH 1.3 反转录 PCR(RT-PCR) 检测 CiGAPDH 基因的时空表达情况 CiGAPDH CiActin, CiGAPDH CiActin CiGAPDH : (CP1)5 - ACGGATCCATGGCTGATAAACATTAC-3 ; : (CP2)5 -ACCTCGAGTCAATGTAAATT TCCATC-3, CiActin (AP1)5 -ATGGCCGAAGACTAACAAGCAG-3 ; : (AP2)5 -TCAGAAGCATTTTCTGTGTACA- 3 Trizol (Takara, ) / RNA, RNA RNA PCR (avian myeloblastosis virus, AMV)Ver.3.0(Takara, ) 10 μl, cdna : min min 95 5 min 5 5 min cdna PCR, AP1 AP2 CiGAPDH, AP1 AP2 CiActin, : CiGAPDH (94 30 s s 72 1 min 10 s) 30 CiActin (94 30 s s 72 1 min 20 s) 30 PCR 1%, CiGAPDH 1.4 CiGAPDH 基因的定向诱变及原核表达载体的构建 CiGAPDH 3 (2 TAA 1 TAG),, CAA CAG, CiGAPDH, GeneTailor TM Site- Directed Mutagenesis System (Invitrogen,, ) ( 1) DNA (Biospin Plasmid DNA Extraction Kit)(BioFlux, ), ptripiex2/cigapdh, PCR, 142 th, PCR, ; PCR E. coli DH5α, DNA McrBC,, BioEdit 12 / 2016 / 40 / 2

3 , 187 th 919 th CiGAPDH,, : 5 -ACGGATCCATGGCTGATAAACATTAC-3 ( BamH I ); 5 - ACCTCGAGTCAATGTAAATTTCCATC-3 ( Xho I ) ptripiex2/cigapdh, PCR, : (94 30 s, s, 72 1 min 10 s) 30 PCR BamH I(Takara, ) Xho I(Takara, ) PCR pgex-4t-3, 1%, 1.. 3, T4, pgex-4t-3, E. coli DH5α, 表 1 CiGAPDH 基因各诱变位点的诱变引物 Tab. 1 Mutagenic primers of CiGAPDH 142 th 5 -ATTGACTATATGACCTATCAATTCAAATAT-3 5 -ATAGGTCATATAGTCAATATCCATAAATG th 5 -AACTGGAAAGGAGAAGTCCAGAAAAAAGGC-3 5 -GACTTCTCCTTTCCAGTTACCATGAATAG th 5 -GATGCAGCTGCAGGAATACAACTTAACAAA-3 5 -TATTCCTGCAGCTGCATCGAAAATAGAAG 重组蛋白 rcigapdh 在大肠杆菌中的诱导表达 E. coli BL21(DE3), 37, LB ( 50 mg/l), r/min, , A ~0.5, - -D- (isopropyl-beta-d-thiogalactopyranoside, IPTG) (, ) 0.5 mmol/l, r/min 4 h r/min 10 min, 4 TNE, 100 mg/l, 1~2 min, 4, r/min 20 min, (Glutathione S-transferase, GST)Sefinose TM Resin (GST Sefinose TM Resin) (, ) rcigapdh GST 2, 5 min, SDS-PAGE( 3%, 12%), G250,, BCA (, ) 1.6 抗 rcigapdh 和 GST 蛋白鼠血清的制备 rcigapdh GST ( GST, GST ) PBS, (Sigma, ) 6 SPF (, ), 80 μg/ 2,, 10 d, rcigapdh GST, 免疫印迹试验 (Western blotting) 鉴定重组蛋白的表达 - (SDS-PAGE), (PVDF), PVDF 3% 37 1 h, ( ), 37 1 h PBS 3 (HRP) IgG (MP biomedicals,, )( ), 37 1 h (DAB, 0.5 mg/ml), Marine Sciences / Vol. 40, No. 2 /

1.8 间接免疫荧光抗体试验 (Indirect Immunofluorescent Antibody Test, IFAT) 检测天然 CiGAPDH 蛋白在幼虫中的定位 0.5% 30 min, (1500 r/min, 5 min),, 100, rcigapdh (1.. 100 IFAT ), GST (1.

4 1.8 间接免疫荧光抗体试验 (Indirect Immunofluorescent Antibody Test, IFAT) 检测天然 CiGAPDH 蛋白在幼虫中的定位 0.5% 30 min, (1500 r/min, 5 min),, 100, rcigapdh ( IFAT ), GST ( IFAT ), 37 1 h PBS 3 Alexa 594 IgG (Invitrogen,, )( IFAT ), 37 1 h 4, (4, 6-diamidino-2-phenylindole, DAPI), PVP (, ), 2 结果及分析 2.1 CiGAPDH 基因的生物信息学分析结果 cdna 1143 bp, 1035 bp, 344, 1 CiGAPDH 37.3 ku, pi , TAA TAG CiGAPDH, CAA CAG CiGAPDH, N NAD + C, 2 PSORT Prediction CiGAPDH CiGAPDH CiGAPDH GAPDH ( 3),, 2.2 CiGAPDH 基因的时空表达情况分析结果 / cdna, CiGAPDH (CP1, CP1), CiGAPDH ( CiActin ), 1035bp ( 4), Fig. 1 1 CiGAPDH The full-length cdna sequence of CiGAPDH and its deduced amino acid sequence Mutation sites are underlined 14 / 2016 / 40 / 2

Fig. 2 2 CiGAPDH The predicted conserved domain of CiGAPGH from C. irritans CiGAPDH, GAPDH 2.3 CiGAPDH 基因的定向诱变 3 11 GAPDH NJ Fig. 3 Neighbor-joining phylogeny tree of 11 GAPDH proteins RnGAPDH.

1); BmGAPDH. (Bombyx mori)gapdh (BAE96011.1); ScGAPDH. (Saccharomyces cerevisiae) GAPDH (EHN01682.1); AtGAPDH. (Arabidopsis thaliana) GAPDH (NP_187062.1); OsGAPDH. (Oryza sativa) GAPDH (AAA82047.

5 Fig. 2 2 CiGAPDH The predicted conserved domain of CiGAPGH from C. irritans CiGAPDH, GAPDH 2.3 CiGAPDH 基因的定向诱变 3 11 GAPDH NJ Fig. 3 Neighbor-joining phylogeny tree of 11 GAPDH proteins RnGAPDH. GAPDH(Rattus norvegicus)gapdh, NP_ ); HsGAPDH. (Homo sapiens) GAPDH(NP_ ); CeGAPDH, (Caenorhabditis elegans)gapdh, (NP_ ); DmGAPDH. (Drosophila mojavensis )GAPDH (XP_ ); BmGAPDH. (Bombyx mori)gapdh (BAE ); ScGAPDH. (Saccharomyces cerevisiae) GAPDH (EHN ); AtGAPDH. (Arabidopsis thaliana) GAPDH (NP_ ); OsGAPDH. (Oryza sativa) GAPDH (AAA ); TtGAPDH. (Tetrahymena thermophila) GAPDH(XP_ ); PmGAPDH. (Perkinsus marinus)gapdh (XP_ ) cdna CiGAPDH 3, BioEdit, T C ptripiex2/cigapdh, PCR, PCR 1%, 1035bp ( 5), Fig. 5 5 CiGAPDH Amplification of the modified ORF of CiGAPDH M DNA ; 1. CiGAPDH M. DNA ladder; 1. CiGAPDH 2.4 原核表达载体的构建 4 CiGAPDH Fig. 4 CiGAPDH expression in C. irritans at different stages M. DNA; 1, 4. PCR ; 2, 5. PCR ; 3, 6. PCR M. DNA ; 1, 4. PCR products of trophonts/protomonts; 2, 5. PCR products of tomonts; 3, 6. PCR products of theronts PCR BamH I Xho I, pgex-4t-3, pgex-4t-3/cigapdh, E. coli DH5α 3 BamH I Xho I, ( 6), CiGAPDH, CiGAPDH pgex-4t-3, pgex-4t-3/cigapdh 2.5 重组蛋白 rcigapdh 的诱导表达 pgex-4t-3/cigapdh E. coli Marine Sciences / Vol. 40, No. 2 /

鼠血清分别做第一抗体进行免疫印迹试验分析(图 8) 结果显示在分子质量约为 37.3 ku 处均有一条特异性反应条带, 说明刺激隐核虫内源性 CiGAPDH 的分子质量是 37.3 ku, 与理论预测值相符; 其次, CiGAPDH 蛋白在刺激隐核虫各期虫体内均有表达; 结果还表明 rcigapdh 蛋白具有很好的免疫原性图6 Fig.

The recombinant plasmid pgex-4t-1/cigapdh was double digested with BamHI and XhoI. BL21(DE3)细胞中, 用 IPTG 诱导表达 4h 后, 超声破碎, 离心后收集上清和沉淀进行检测, 并用 GST 纯化胶对上清中的表达产物进行纯化 SDS-PAGE 结果显示: 在分子质量约为 63.

irritans with mouse antiserum against rcigapdh 与 GST-CiGAPDH 融合蛋白的预期大小一致 A. 氨基黑 10B 染色; B. 一抗为 rcigapdh 免疫鼠血清; C. 一抗 (CiGAPDH 的分子质量 37.

6 鼠血清分别做第一抗体进行免疫印迹试验分析(图 8) 结果显示在分子质量约为 37.3 ku 处均有一条特异性反应条带, 说明刺激隐核虫内源性 CiGAPDH 的分子质量是 37.3 ku, 与理论预测值相符; 其次, CiGAPDH 蛋白在刺激隐核虫各期虫体内均有表达; 结果还表明 rcigapdh 蛋白具有很好的免疫原性图6 Fig. 6 重组质粒 pgex-4t-3/ CiGAPDH 的双酶切鉴定 Identification of the recombinant plasmid pgex4t-1/cigapdh by double restriction digestion M. DNA 分子质量标准; 1. 重组质粒经 BamH I 和 Xho I 双酶切后的结果 M. DNA ladder: 1. The recombinant plasmid pgex-4t-1/cigapdh was double digested with BamHI and XhoI. BL21(DE3)细胞中, 用 IPTG 诱导表达 4h 后, 超声破碎, 离心后收集上清和沉淀进行检测, 并用 GST 纯化胶对上清中的表达产物进行纯化 SDS-PAGE 结果显示: 在分子质量约为 63.3 ku 处有一明显条带, 图8 免疫印迹试验分析 rcigapdh 蛋白免疫鼠血清与刺 Fig. 8 激隐核虫天然 CiGAPDH 蛋白的抗原抗体反应 Western blot analysis of native CiGAPDH from C. irritans with mouse antiserum against rcigapdh 与 GST-CiGAPDH 融合蛋白的预期大小一致 A. 氨基黑 10B 染色; B. 一抗为 rcigapdh 免疫鼠血清; C. 一抗 (CiGAPDH 的分子质量 37.3 ku, GST 为 26 ku), 在可为 GST 免疫鼠血清溶相和不溶相中均有表达, 说明 CiGAPDH 基因在大 M. 蛋白质分子质量标准; 1-3. 抗原分别为刺激隐核虫滋养体肠杆菌中成功表达(图 7) 包囊及幼虫裂解液; 4. 抗原为 GST A. Native CiGAPDH protein staining by amino black 10B, B. Native CiGAPDH reacted with antiserum against rcigapdh, C. Reaction with antiserum against GST M. markers: 1 3. Lysates of C. irritans trophonts, tomonts, and theronts, respectively, and 4. GST used as a control. 2.7 天然 CiGAPDH 蛋白在刺激隐核虫幼虫中的定位以抗 rcigapdh 蛋白免疫鼠血清为第一抗体 Alexa 594 标记的羊抗鼠 IgG 抗体为第二抗体, 同时以抗 GST 鼠血清作对照, 运用 IFAT 法检测天然图 7 大肠杆菌表达重组蛋白 rcigapdh 的 SDS-PAGE 分析结果 Fig. 7 Prokaryotic expression of recombinant CiGAPDH analyzed by SDS-PAGE CiGAPDH 蛋白在刺激隐核虫幼虫中的定位(图 9) 结果显示天然 CiGAPDH 蛋白主要分布在刺激隐核虫幼虫的细胞质内, 且在胞口位置分布最多, 这与 CiGAPDH 蛋白进行亚细胞定位预测的结果是一 M. 蛋白质分子质量标准; 1. 不溶性组分; 2. 可溶性组分; 3. 纯致的化后的重组蛋白; 4. GST M. Proteins with standard molecular mass: 1. Insoluble fractions, 2. Soluble fractions, 3. Purified rcigapdh, and 4. GST 类养殖业, 被农业部列入二级动物疫病名录 [ ], 讨论刺激隐核虫病严重危害我国东南沿海的经济鱼重组蛋白 rcigapdh 的免疫印迹试验鉴定结果对该病防治的重要性可见一斑从分子生物学水平刺激隐核虫滋养体/前包囊包囊和幼虫全虫蛋深入了解其病原生物学将对阻断该病的传播具有积白经 SDS-PAGE 后, 电转到 PVDF 膜上, 并以 GST 极的意义本实验通过对克隆自刺激隐核虫的作对照, 用抗 rcigapdh 免疫鼠血清和抗 GST 免疫 GAPDH 同源基因(CiGAPDH)的序列分析对重组蛋 16 海洋科学 / 2016 年 / 第 40 卷 / 第 2 期

7 9 CiGAPDH Fig. 9 Localization of native CiGAPDH in the theronts by IFAT A. DAPI ; B. rcigapdh ( ) GST ( ) ; C. A B ; D. A. Nucleus, B. Immunofluorescence figure with antiserum against rcigapdh (left) or GST (right), C. A merged image of Figs. A and B, D. Bright-field image,, GAPDH, : CiGAPDH, N NAD + C, ; GAPDH,, ;, CiGAPDH, ; ; CiGAPDH, GAPDH ; CiGAPDH : CiGAPDH 3 (2 TAA 1 TAG, ) [19], PCR, CAA CAG,, rcigapdh SPF, rcigapdh, : rcigapdh, rcigapdh CiGAPDH, GAPDH,, [20-25], CiGAPDH, : [1] Wright A D, ColorniA. Taxonomic re-assignement of Cryptocaryon irritants, a marine fish parasite. European journal of protistology[j]. 2002, 37(4): [2] Sikama Y. Preliminary report on the white spot disease in marine fishes [J]. Suisan-Gakukai-Ho, 1937, 7: [3] Colorni A, Burgess P. Cryptocaryon irritans Brown 1951, the cause of white spot disease in marine fish: an update [J]. Aquarium Sciences and Conservation, 1997, 1(4): [4] Luo X C, Xie M Q, Zhu X Q, et a1. Some characteristics of host parasite relationship for Cryptocaryon irritans isolated from South China [J]. Parasitol Res, 2008, 102 (6): [5] Burgess P, Matthews R. Fish host range of seven isolates of Cryptocaryon irritans (Ciliophora) [J]. Journal of fish biology, 1995, 46(4): [6] Diggles B K, Adlard R D. Intraspecific variation in Cryptocaryon irritans [J]. J Eukaryot Microbiol, 1997, 44(1): Marine Sciences / Vol. 40, No. 2 /

8 [7] Sikama Y. On a new species of Ichthyophthirius found in marine fishes [J]. Science Report of the Yokosuka City Museum, 1961, 6: [8] Hirazawa N, Goto T, Shirasu K. Killing effect of various treatments on the monogenean Heterobothrium okamotoi eggs and oncomiracidia and the ciliate Cryptocaryon irritans cysts and theronts [J]. Aquaculture, 2003, 223(1-4): [9] Kawano F, Hirazawa N. Antiparasitic effect of in-feed inhibitors of folic acid synthesis and dihydrofolate reductase against ciliate Cryptocaryon irritans infection in the red sea bream Pagms major and against ciliate Ichthyoph thirius muhifiliis infection in black pop-eyed goldfish Carassius aurattas [J]. Aquaculture, 2012, 330/331/332/333: 1-7. [10],,. [M]. 3. :, 2002: Wang Jingyan, Zhu Senggeng, Xu Changfa, et a1. Biological Chemistry, 3rd ed[m]. Bei Jing: Higher Education Press, 2002: [11] Viscogliosi E, Miiller M. Phylogenetic relationships of the glycolytic enzyme, glyceraldehyde-3 phosphate dehydrogenase, from parabasalid flagellates[j]. Mol Evol, 1998, 47(2): [12] Yan H, Lou M F, Fernando M R, et a1. Thioredoxin, thioredoxin reductase, and alpha erystallin revive inactivated yceraldehyde-3-phosphate dehydro-genase in human aged and cataract lens extracts[j]. Mol Vis, 2006, 12: [13] Vollberg T M, Siegler K M, Cool B L, et al. Isolation and characterization of the human uracil DNA gycosylase gene [J]. Proceedings of the National Academy of Sciences of the United States of America, 1989, 86(22): [14] Baxi M D, Vishwanatha J K. Uracil DNA-glycosylase/ glyceraldehyde-3-phosphate dehydrogenase is an Ap4A binding protein [J]. Biochemistry, 1995, 34(30): [15] Tarze A, Deniaud A, Le Bras Met al. GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization[j]. Oncogene, 2007, 26(18): [16] Morero R D, Vinals A L, Bloj B, et al. Fusion of phospholipid vesicles induced by muscle glyceraldehyde- 3-phosphate dehydrogenase in the absence of calcium [J]. Biochemistry, 1985, 24(8): [17] Yang T B, Chen A P, Chen W, et al. Parasitic diseases of cultured marine finfishes and their surveillance in China [J]. Parassitologia, 2007, 49(3): [18]. [J]., 2009, 36(1): 4-7. Su Yuezhong. Prevention and cure of fish disease Surveys and control countermeasures of diseases of Cryptocaryon irritans in marine cage cultured fishes in the main cultured areas of Fuiian province[j]. Fish Sci Technol Inform, 2009, 36 (1): 4-7. [19] Salim H M, Ring K L, Cavalcanti A R. Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia [J]. Protist, 2008, 159(2): [20] Morero R D, Vinals A L, Bloj B, et al. Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3- phosphate dehydrogenase in the absence of calcium [J]. Biochemistry, 1985, 24(8): [21] Robbins A R, Ward R D, Oliver C. A mutation in glyceraldehyde-3-phosphate dehydrogenase alters endocytosis in CHO cells [J]. J Cell Biol, 1995, 130(5): [22] Tisdale E J. Glyceraldehyde-3-phosphate dehydrogenase is required for vesicular transport in the early secretory pathway [J]. J Biol Chem, 2001, 276(4): [23] Glaser P E, Han X, Gross R W. Tubulin is the endogenous inhibitor of the glyceraldehyde-3-phosphate dehydrogenase isoform that catalyzes membrane fusion: Implications for the coordinated regulation of glycolysis and membrane fusion [J]. Proc Natl Acad Sci USA, 2002, 99(22): [24] Bryksin A V, Laktionov P P. Role of glyceraldehyde-3- phosphate dehydrogenase in vesicular transport from Golgi apparatus to endoplasmic reticulum [J]. Biochemistry- Moscow, 2008, 73(6): [25] Tisdale E J, Kelly C, Artalejo C R. Glyceraldehyde-3- phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity [J]. J Biol Chem, 2004, 279(52): / 2016 / 40 / 2

9 Molecular characterization of a Glyceraldehyde-3-phosphate dehydrogenase from Cryptocaryon irritans FU Guo-liang 1, 2, SHAN Jin-hong 1, HU Yan-hong 1, XIE Jin-zhu 1, YAN Yan-hua 1, 2, HUANG Xiao-hong 1 (1. Fujian Key Laboratory of Developmental and Neural biology, College of Life Science, Fujian Normal University, Fuzhou , China; 2. Dongxiang No.1 High School of Jiangxi, Dongxiang , China) Received: Jul., 7, 2015 Key words: Cryptocaryon irritans; glyceraldehyde 3-phosphate dehydrogenase; prokaryotic expression; location Abstract: A GAPDH gene (CiGAPDH) was cloned from the cdna library of Cryptocaryon irritans trophonts. After modification, the non-universal genetic codons in the open reading frame (ORF) of CiGAPDH were inserted into plasmid pgex-4t-3 to construct the prokaryotic expression plasmid pgex-4t-3/cigapdh. The recombinant plasmids were transformed to Escherichia coli BL21 (DE3) cells, which were then induced to express the foreign gene by the addition of isopropyl-beta-d-thiogalactopyranoside. SDS-PAGE results showed that rcigapdh was successfully expressed in E. coli cells. The results from Western blot analysis showed that antiserum against rci- GAPDH recognized the native CiGAPDH protein from different stages of C. irritans, the molecular mass of which was 37.3 ku, in agreement with the calculated mass. Localization of native CiGAPDH protein in theronts was detected using an indirect immunofluorescence cell imaging technique. The results showed that the native CiGAPDH protein was mainly distributed in the cytoplasm of C. irritans theronts, with notable accumulation around the cytostomes. Further study on the roles of CiGAPDH in the parasite development and infection is expected to provide important information to identify potential drug targets for control of cryptocaryosis. ( 本文编辑 : 梁德海 ) Marine Sciences / Vol. 40, No. 2 /

Invitrogen 1.4 pet-gr, ICE-LGR, PCR TA, pmd18-t/gr pet-21a(+) (Nde I Xho I), E. coli DH-5,,,, pet-gr GR ORF cdna, GR, : PU: 5 -GGAATTCCAT

Invitrogen 1.4 pet-gr, ICE-LGR, PCR TA, pmd18-t/gr pet-21a(+) (Nde I Xho I), E. coli DH-5,,,, pet-gr GR ORF cdna, GR, : PU: 5 -GGAATTCCAT 42 6 Vol.42, No.6 2011 11 OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2011 南极衣藻 (Chlamydomonas sp. ICE-L) 谷胱 * 甘肽还原酶基因的原核表达及其条件优化 1 1 燏 1 1 2 (1. 524025; 2. 266061) RT-PCR GR ORF cdna, ; IPTG,, pet-gr BL21,