正常鲤外周血白细胞cDNA文库的构建
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中国人民解放军军需大学动物科技系

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国家自然科学基金


Construction of cDNA library of peripheral blood leucocytes in normal Cyprinus carpio L.
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Faculty of Animal Science and Veterinary Medicine, Quartermaster University of PLA, Changchun130062, China

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    Leukocytes are key cells for the specific and nonspecific immune systems , it expresses different kinds of biodefense2related peptides and proteins including specific and nonspecific antimicrobial agents , and activators and regulators of the immune system. However,we know very little about the cellular interactions that initiate and control the adaptive immune response in fish. In order to clone and study immune2related and biodefense2related genes in carp ( Cyprinus carpio L. ) leucocytes , the cDNA library of carp leucocytes was constructed. First , the carp leucocytes were isolated from the peripheral blood of normal carp , total RNA of leucocytes was extracted and from 2 ?108 leucocytes cells , and the output was 216μg , ODA260/ 280 > 1. 8 , mRNA was isolated with a column of oligo (dT) cellulose. Second, single2strand cDNA and double2strand cDNA were synthesized from 5μg mRNA using Stratagene HybriZAP22. 1 XR cDNA synthesis kit , then ligated to EcoR I adapters , size fractionating with CHROMA Spin2400 column. Finally cDNA were ligated into HybriZAP22. 1 vector and packaged in vitro. The obtained carp leucocyte cDNA library contains 5. 79 ?106 recombinants , the percentage of vectors with inserts was 99. 4 % and the average inserts size were between 0. 4 ?103 -3.0 ?103bp. After amplification , the titer of amplified library was 4. 22 ?109pfu?mL -1 . This normal carp leucocytes cDNA library gives an ideal basis for further study of carp immune system.

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卢强.正常鲤外周血白细胞cDNA文库的构建[J].水产学报,2004,28(5):

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  • 收稿日期:2014-03-14
  • 最后修改日期:2014-03-14
  • 录用日期:2014-03-14
  • 在线发布日期: 2014-03-14
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