摘要
目的构建海南产桶形芋螺毒管cDNA文库,为桶形芋螺毒素基因资源的永续保存和新型药物的研发提供依据。方法以总RNA抽提试剂盒,从桶形芋螺毒管中提取总RNA,采用SMART技术,LD-PCR扩增获得双链cDNA,经SfiⅠ酶切和CHROMA SPIN-400柱分级分离后,将500bp以上的片段与载体pDNR-LIB连接,通过电穿孔转入E.coli JM109,获得原始文库;随机挑取单菌落,经菌液PCR鉴定重组率及插入片段大小,最后扩增文库。结果经鉴定,所得文库的容量约为5.0×106个克隆,原始文库的平均滴度为5.01×108,插入片段平均长度为1.0kb,文库的重组率达到92%。结论所构建的文库是合格的,为新型芋螺毒素的发现和研究利用提供了依据。
Objective To conserve the precious conotoxin gene resource of Conus betulinus Linnaeus collected from Hainan, a cDNA library of the venom duct C. betulinus was constructed, which would provide the biomaterial for investigating and developing the original marine conopeptide drugs. Methods Total RNA was extracted from the venom ducts C. betulinus by Total RNA Isolation Kit, and the long-distance PCR （LD-PCR） method was used to amplify double strand eDNA based on the SMART techniques. After digestion with SfiI and size fractionation by Chromaspin-400 columns, cDNAs （〉500bp） were ligated to dephosphorylated pDNR-LIB vector digested by Sfi I. Then, the recombinant plasmids were transformed into E. colt JM109 through electroporation. The original library was generated through pooling the desired transformation mixtures. Clones were picked randomly from the library for screening the size of the cDNA inserts through Bacteria Liquid PCR. The last step was the library amplification. Results The original constructed eDNA library contained 5.0 × 10^6 independent clones. The titer of the cDNA library was about 5.01 × 10^8 cfu/mL, with the percentage of recombinant clones about 92 %. The average size of the inserts was about 1 Kb. Conclusion The constructed eDNA library meets the requirement of a standard eDNA library. It could be a strong basis for subsequent work of finding and researching some kinds of new conotoxins.
出处
《中国海洋药物》
CAS
CSCD
2009年第3期1-6,共6页
Chinese Journal of Marine Drugs
基金
国家自然科学基金(30560184
30860368)
国家863计划(2007AA02Z114)
海南省重点学科建设项目专项
海南省教育厅高等学校科研项目(Hjkj200719)
海南省自然科学基金(80618)
关键词
桶形芋螺
毒管
CDNA文库
Conusbetulinus Linnaeus
venom ducts
cDNA library
