Leukocytes are key cells for the specific and nonspecific immune systems, it expresses different kinds of biodefense-related peptides and proteins including specific and nonspecific antimicrobial agents, and activator...Leukocytes are key cells for the specific and nonspecific immune systems, it expresses different kinds of biodefense-related 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 immune-related and biodefense-related 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, single-strand cDNA and double-strand cDNA were synthesized from 5μg mRNA using Stratagene HybriZAP-2.1 XR cDNA synthesis kit, then ligated to EcoR I adapters, size fractionating with CHROMA Spin-400 column. Finally cDNA were ligated into HybriZAP-2.1 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.展开更多
文摘Leukocytes are key cells for the specific and nonspecific immune systems, it expresses different kinds of biodefense-related 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 immune-related and biodefense-related 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, single-strand cDNA and double-strand cDNA were synthesized from 5μg mRNA using Stratagene HybriZAP-2.1 XR cDNA synthesis kit, then ligated to EcoR I adapters, size fractionating with CHROMA Spin-400 column. Finally cDNA were ligated into HybriZAP-2.1 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.
