摘要
mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyAT-tract System 1000 Kit. By using mRNA as template, double - strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR l/Xho Ⅰ, and the recombinant DNA was in vitro packaged into lambda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XL1 - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 10~5pfu in capacity and its recombination ratio is higher than 99 % . The size of the inserted cDNAs was determined by EcoR l/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus monodon hepatopancreas and is available for screening and expression of shrimp genes.
mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyAT-tract System 1000 Kit. By using mRNA as template, double - strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR l/Xho Ⅰ, and the recombinant DNA was in vitro packaged into lambda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XL1 - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 10~5pfu in capacity and its recombination ratio is higher than 99 % . The size of the inserted cDNAs was determined by EcoR l/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus monodon hepatopancreas and is available for screening and expression of shrimp genes.
基金
This study was supported by the Ocean Science
Technology Fundation of State Oceanic Administration
Ocean "863" Project under contract No. 2001AA621130.
