| 摘要: |
| 以本实验室构建的鸡致病性大肠杆菌诱导家蝇幼虫抑制性消减杂交文库（SSH）为基础，对家蝇幼虫溶菌酶II基因（MdL-II)进行克隆，得到全长为532 bp的cDNA片段，其开放阅读框（ORF）与GenBank中登录号为HQ897688.1的MdL-II基因全序列同源性为95%。将MdL-II ORF序列插入到pET-32a(+)表达载体中成功构建重组质粒，其表达产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，结果显示约在34 ku处出现表达条带且与目的蛋白大小相符。当诱导温度为37 ℃，IPTG浓度为0.6 mmol/L时可获得大量可溶性Trx-MdL-II融合蛋白，纯化融合蛋白并进一步检测该蛋白的抑菌活性，结果表明融合蛋白对大肠杆菌和链球菌均有抑菌作用，且对大肠杆菌的抑菌作用相对较强。 |
| 关键词: 家蝇幼虫 家蝇溶菌酶II基因 克隆表达 蛋白纯化 |
| DOI： |
| 投稿时间：2014-11-25修订日期：2015-01-05 |
| 基金项目:教育部新世纪优秀人才项目(NCET-10-0174)；吉林省世行贷款农产品质量安全项目(2011-Y05)；吉林省科技厅项目(20111820) |
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| Cloning and Prokaryotic Expression of the Lysozyme II Gene from Musca domestica Larvae |
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| (College of Animal Science and Technology,Jilin Agriculural University,Changchun) |
| Abstract: |
| The experiment was based on the suppression subtractive hybridization library (SSH) induced by Pathogenic Escherichia coli of chicken which constructed by Animal Pharmacology and Toxicology Laboratory. Amplify Md-lysozyme II gene’sfull-length cDNA, then sequencing analysis showed that the full-length cDNA of Md-lysozyme II gene was 532 bp, in size,the homology of the open reading frame cDNA sequence with the Md-lysozyme II(HQ897688.1) from GenBank is up to 95%. The Md-lysozyme II gene was ligated into expression vector pET-32a( ). The recombinant protein was verified by SDS-PAGE,the results showed that the recombinant protein was about 34 ku and consistent with the size of the target protein.When the induction temperature was 37 ℃ and the concentration of IPTG was 0.6 mmol/L we could get a large amount of soluble Trx-MdL-II recombinant protein..In order to obtain high purity recombinant protein we used nickel ion affinity chromatography to purify Trx-MdL-II recombinant protein. While the recombinant protein could inhibit the growth of Escherichia coli and Streptococcus, the inhibitory effect on Escherichia coli was relatively stronger than on Streptococcus. |
| Key words: Musca domestica larvae Md-lysozyme II cloning and expression protein purification |
