Construction Of CDNA Phage Library Of The Venom Duct Of Conus Lividus By Switching Mechanism At5’ End RNA Transcription Technology And The Cloning Of The Novel Conotoxin Genes

Conotoxins come from the venom of gastropod mollusk cone shells which is sarcophagy and distribute in the tropical waters of the Indian and Pacific Oceans. Conotoxin is a kind of cysteine-rich neuropeptide, which consists of7-40amino acid residues and has construction of disulfide bonds. Conotoxins selectively act on various kinds of receptors of neurotransmitters, ion channels and neurokinins, so conotoxins can be used as research tools of ion channels and membrane receptors. Meanwhile they have the potential to be new drugs or leading compounds. They have become a research hotspot of neurobiology, biochemistry, pharmacology, pharmaceutical chemistry and militarybranch.Cruz et al isolated the first conotoxin from the venom of Conus geographus in1978. Up to now there are hundreds of conotoxins have been isolated and identified, but the procedures of collection, extraction, isolation and purification of conuses are time consuming, strenuous and low yield. The collection of the conuses is influenced by its resources, besides some samples are difficult to be obtained. As the development of the biotechnology, to obtain various kinds of conotoxins using the method of genetic engineering is the major leading of the research in future. It solves the problem of the source of conuses by constructing cDNA library, and the prerequisite of all the work is the discovery of sundry conotoxin genes.Total RNA was extracted from the venom duct of Conus lividus. Single-(ss-cDNA)and double-stranded cDNA(ds-cDNA) were synthesized through reverse transcription polymerase chain reaction(RT-PCR) and long distance polymerase chain reaction(LD-PCR). CDNA fragments, after digestion by proteinase K and Sfi Ⅰ enzyme and fractionation by CHROMA SPIN-400, were recombined to λTripIE×2phage vector. The recombined cDNA was packaged with GigapackⅢ Gold packing extract in vitro. A small portion of packing phage was used to infect Escherichia XL1-blue for titration and determination of the percentage of recombinant clones. The remainder portion of packing phage was used to infect Escherichia XL1-blue for amplification,and then the titer of the amplified library was detected. The size of the inserted cDNA was identified by PCR. The constructed cDNA phage library of the venom duct of Conus lividus consisted of1.47×106pfu/ml independent clones with a recombination rate of90.72%. After amplification, the titer of the amplified library was4.07×108pfu/ml. The lengths of the inserted cDNA fragments ranged from200to1200bp. A high-quality cDNA phage library of the venom duct of Conus lividus was successfully constructed. This is an essential step for screening and identifying new conotoxin genes of Conus lividus.Although the cDNA phage library has the advantage of high quality, big capacity, good representation, being appropriate for long-time conservation and so on, the work of the phage plating is so laborious, low-level efficiency and the method of the gene screeing is limited. In accordance with the theory of the conversion of a λTripIEx2clone to a pTripIE×2plasmid when the recombinant phage is transduced into a E.coli BM25.8bacterial host in which cre recombinase is being expressed, the cDNA phage library is transformed into a cDNA plasmid library. The cDNA plasmid library actualizes easy clone of the genes and breaks through the limitation of the gene screeing. It has broad prospects on the mass screeing of the novel genes.