蟾蜍(Bufo japonicus)细胞溶素基因筛选用探针的制备

PREPARATION OF SCREENING PROBES FOR LYSIN GENE FROM BUFO JAPONICUS

利用阴离子交换、凝胶过滤和高压液相柱层析对细胞溶素基因进行了纯化，获得了纯度极高的酶样品．经ｌｙｓｉｌ 内肽酶进行定点酶切后，利用高压液相柱层析纯化出了该酶的多个短肽片段．其中，经测序获得了３ 个短肽片段的氨基酸序列．根据两栖动物中密码子的使用频率，可推测出其相应ｃ Ｄ Ｎ Ａ 的核苷酸序列，进而制备出了３ 个ｃ Ｄ Ｎ Ａ 探针．利用这些合成的探针，便可从精子ｃ Ｄ Ｎ Ａ 文库中筛选细胞溶素基因．

Lysin gene structure and its gene express were studied and regulated during fertilization in Bufojaponicus . Pure lysin was obtained via anion exchange ,gel filtration ,and H P L Cchromatography . Manyshort peptides were acquired in H P L C purification of lysin digested by lysil endopeptidase . Amongthese ,the amino acid sequence of three peptides were obtained . Based on the emerging frequency ofcodons in amphibian ,their nucleotide sequences were predicted . Therefore ,three c D N Aprobes was pro duced by P C R method . With these specific c D N Aprobes ,lysin gene could be screened out from toadsperm c D N Alibrary .