| Luo Tian,Xu Xun. 2002. Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon. Acta Oceanologica Sinica, (3):437－443 |
| Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon |
| Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon |
| Received:December 17, 2001 Revised:January 20, 2002 |
| DOI： |
| Key words:Penaeus monodon cDNA expression library hepatopancreas |
| 中文关键词: Penaeus monodon cDNA expression library hepatopancreas |
| 基金项目:This study was supported by the Ocean Science; Technology Fundation of State Oceanic Administration; Ocean "863" Project under contract No. 2001AA621130. |
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| Abstract: |
| mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyATtract 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 I/Xho I, and the recombinant DNA was in vitro packaged into larnbda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XLl-Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2×105pfu in capacity and its recombination ratio is higher than 99%. The size of the inserted cDNAs was determined by EcoR I/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 modon hepatopancreas and is available for screening and expression of shrimp genes. |
| 中文摘要: |
| mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyATtract 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 I/Xho I, and the recombinant DNA was in vitro packaged into larnbda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XLl-Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2×105pfu in capacity and its recombination ratio is higher than 99%. The size of the inserted cDNAs was determined by EcoR I/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 modon hepatopancreas and is available for screening and expression of shrimp genes. |
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