鱼类培养细胞抗病毒基因差减cDNA文库的构建
CONSTRUCTION OF ANTIVIRAL SUBTRACTIVE cDNA LIBRARY OF CULTURED FISH CELLS
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摘要: 紫外线灭活的草鱼出血病病毒(GCHV)能诱导鲫囊胚培养细胞(CAB)产生高滴度的干扰素,从而诱导宿主细胞基因表达的改变并处于抗病毒状态.提取灭活病毒诱导未经病毒诱导的CAB细胞mRNA,利用抑制性差减杂交技术,成功构建了鱼类培养细胞抗病毒基因差减cDNA文库.以鲫管家基因α-tubulin和β-actin作为差减指标,检测差减cDNA文库的差减效率分别高达215和27倍,表明经过病毒诱导后的细胞中,某些差异表达基因的富集效率也接近215倍.鱼类抗病毒基因差减cDNA文库的建立对快速分离、克隆鱼类抗病毒相关基因和认识鱼类细胞抗病毒免疫的分子机理有重要意义.Abstract: The vertebrate interferon (IFN) system provides an initial line of defense against viral infection by inducing the synthesis of proteins that inhibit virus replication, but the normal cells do not, as a rule, contain or synthesize IFN. UV-inactivated GCHV is very effective for inducing blastulae embryonic cells of crucian carp (Carassius auratus L.)(CAB) to secrete interferon, which in turn act in both autocrine and paracrine fashion to induce the expressions of interferon responsive genes including antiviral genes, and finally contribute to form antiviral states in host cells. However, to date little was known about these proteins and genes. In order to analyze the change of gene expressions and isolate these related genes induced or activated by virus infection in CAB cells, an antiviral subtractive cDNA library of CAB was constructed by using Suppression Subtractive Hybridization (SSH) techniques. Typically, GCHV was purified and inactivated under UV irradiation for 5 min, and then CAB cells were treated with UV-inactivated GCHV. The mRNAs were extracted from virally infected CAB cells and mock-infected cells, respectively, and used for constructing subtractive cDNA library. In the present paper, the quality of cellular RNAs and adaptor ligation efficiency were analyzed, and two housekeeping genes, α-tubulin and β-actin, were used to estimate the efficiency of subtractive cDNA. In the antiviral subtractive cDNA library, the two housekeeping genes were subtracted very efficiently at appropriate 215 and 27 folds, respectively, indicating that some differentially expressed genes including antiviral genes induced by viral infection were also enriched at the same folds. The subtractive cDNAs were ligated into the pGEM-T EASY vector (Promega) and following transfection into competent E.coli cells. 16 colonies were randomly selected from the plasmid library, and PCR anlysis showed that the inserted cDNA fragments were between 0.2—2kb in length. The results suggected that the antiviral subtractive cDNA library is very successful, which will be essential for rapid isolation of fish antiviral genes and helpful for elucidation of molecular mechanism of fish innate defense against virul infections in the future.
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Keywords:
- Fish /
- Grass carp hemorrhage virus /
- Antiviral related gene /
- Induction /
- SSH
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Fish Shellfish Immunol,1993,3(2):89-96[17] Trobridge G D, Leong J C. Characterization of a rainbow trout Mx gene [J]. J interferon Cytokine Res,1995,15:691-702[18] Trobridge G D, Chiou P P, Leong J C. Cloning of the rainbow trout (Oncrohunchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells [J]. J virol,1997,71:5304-5311[19] Robertsen B, Trobridge G D, Leong J. Molecular cloning double-stranded RNA inducible Mx genes from Atlantic salmon (Salmo salar L.)[J]. Dev Comp Immunol, 1997,21:397-412[20] Lee J Y, Hirono I, Aoki T. Cloning and analysis of expression of Mx cDNA in Japanese flounder, paralichthys olivaceus [J]. Dev Comp Immunol,2000,24(4):407-415 Zhang Y B,Yu X M. Progresses in studies on fish interferon [J]. Journal of Fishery Sciences of China. 2000,7(3):97-101[张义兵,俞小牧. 鱼类干扰素的研究进展[J]. 中国水产科学. 2000,7(3):97-101][2] Gudding R, Lillehaug A, Evensen . Recent developments in fish vaccinology [J]. Veterinary Immunology and Immunopathology. 1999,72:203-312[3] Xie J, Wen J J, Chen B, et al. Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps [J]. Gene,2001,271:109-116[4] Shi Y H, Liu J, Xia J H, et al. Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp [J]. Cell Research, 2002, 12(2):133-142[5] Fan L C, Yang S T, Gui J F. Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp [J]. Cell research,2001,11(1):17-27[6] Wang T H, Zhang Y B, Li G Q, et al. Interferon induction in cultured fish cells [J]. Chinese Journal of Viology, 1999, 15(1):43-49[王铁辉,张义兵,李戈强等. 鱼类培养细胞干扰素的诱导[J]. 病毒学报,1999,15(1):43-49][7] Jia F J, Wang T H, Zhang Y B, et al. Preliminary study on prevention and treatment to grass carp hemorrhagia with interferon [J].Fisheries Scrience, 2000,19(4):1-4[贾方钧,王铁辉,张义兵等. 干扰素防治草鱼出血病的效果初探[J]. 水产科学,2000,19(4):1-4][8] Zhang Y B, Wang T H, Li G Q, Jia F J, Yu X M. Induction and partial characterization of CAB cell (Blastulae Embryonic Cell Line of Crucian Carp) interferon [J]. Virologica Sinica, 2000,15:163～169[张义兵,王铁辉,李戈强等. 鲫囊胚细胞干扰素的诱导及部分特性[J].中国病毒学. 2000,15(2):163-169][9] Chen M R, Chen H X, Yi Y L. The establishment of a heteroploid line from crucian carp and its biological characteristics [J]. Journal of Fisheries of China, 1985,9(2):121-130[陈敏容,陈宏溪,易咏兰. 鲫鱼异倍体细胞系的建立及生物学特性[J]. 水产学报,1985,9(2):121-130][10] Zuo W G, Qian H X, Xu Y F, et al. A cell line derived from the kidney of grass carp [J]. Journal of Fisheries of China. 1984,10:11-17[左文功,钱华鑫,许映芳等. 草鱼肾脏组织细胞系CIK的建立[J].淡水渔业,1984,(2):38-39][11] Ke L H, Fang Q, Cai Y Q. Characteristics of a new isolation of hemorrhagic virus of grass carp [J]. Acta Hydrobiology Sinica. 1990,14(2):153-159[柯丽华,方勤,蔡宜权. 一株新的草鱼出血病病毒分离物的特性[J]. 水生生物学报,1990,14(2):153-159]［12］Zhang Y B, Gui J F. Interferon-inducible Mx proteins in fish [J].Virologica Scinica,2001,16(4):291-298[张义兵,桂建芳. 干扰素诱导的鱼类Mx蛋白[J]. 中国病毒学. 2001,16(4):291-298][13] Ellis A E. Innate host defense mechanisms of fish against viruses and bacteria [J]. Developmental and comparative Immunology. 2001,25:827-839[14] Stark G R, Kerr I M, Williams B R, et al. How cells respond to interferons [J]. Ann Biochem. 1998,67:227-264[15] Eaton W D, Antiviral activity in four species of salmonids following exposure to poly inosinic:cytidylic acid [J]. Dis Aquat Org, 1990,9:193-198[16] Pinto R M, Jofre J, Bosch A. Interferon-like activity in sea bass affected by viral erythrocytic infection [J]. Fish Shellfish Immunol,1993,3(2):89-96[17] Trobridge G D, Leong J C. Characterization of a rainbow trout Mx gene [J]. J interferon Cytokine Res,1995,15:691-702[18] Trobridge G D, Chiou P P, Leong J C. Cloning of the rainbow trout (Oncrohunchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells [J]. J virol,1997,71:5304-5311[19] Robertsen B, Trobridge G D, Leong J. Molecular cloning double-stranded RNA inducible Mx genes from Atlantic salmon (Salmo salar L.)[J]. Dev Comp Immunol, 1997,21:397-412[20] Lee J Y, Hirono I, Aoki T. Cloning and analysis of expression of Mx cDNA in Japanese flounder, paralichthys olivaceus [J]. Dev Comp Immunol,2000,24(4):407-415
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