【作者】 施婉君；

【导师】 程家安； 张传溪；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2003， 博士

【摘要】 蜂毒溶血肽(Melittin)是意大利蜜蜂蜂毒的最主要成分，约占蜂毒干重的40％～50％。它具有很强的生物学活性，可引起红细胞裂解、脂质体释放标记离子以及肥大细胞释放组胺，同时它对细胞膜也具有很强的表面活性，其透过卵磷脂膜和混合脂膜的速度为任何表面活性剂所不及。因而，它是目前医药、生物工程及农业上家畜疾病的治疗和植物保护方面研究的重要原料。 首先本研究分别从中华蜜蜂、意大利蜜蜂工蜂和雌性亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂毒腺中抽提总RNA，通过RT-PCR方法扩增得到2种蜜蜂和4种胡蜂蜂毒前溶血肽原的cDNA，再将扩增产物克隆到pGEM-T easy vector上。测序结果表明：扩增得到的片段长度均为213 bp，系蜂毒前溶血肽原编码区的cDNA，共编码70个氨基酸，包括由21个氨基酸组成的信号肽和49个氨基酸组成的溶血肽原，溶血肽位于其COOH端(第44～70残基)。经序列比较，2种蜜蜂和4种胡蜂蜂毒前溶血肽原之间的氨基酸序列同源性都超过92％。中华蜜蜂、意大利蜜蜂和亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂各自与东方蜜蜂印度亚种蜂毒前溶血肽原氨基酸序列的同源性分别为97.2％、100％、93％、100％、97.2％、97.2％。结果表明，蜂毒前溶血肽原一级结构序列具很高的保守性，尽管胡蜂和蜜蜂属于膜翅目不同的总科，但它们的前溶血肽原基因却非常相似。以前胡蜂总科被认为不存在溶血肽基因，本研究首次揭示了胡蜂总科存在与蜜蜂相似的溶血肽基因。本文报道的中华蜜蜂、亚非马蜂、额斑黄胡蜂、墨胸胡蜂和大胡蜂的前溶血肽原基因都已作为新的基因序列在GenBank登录，分别为：Apis cerana cerana prepromelittin mRNA(登录号AF487907)、Polistes hebraeus prepromelittin mRNA(登录号AF487909)、Vespula maculifrons prepromelittin mRNA(登录号AF487911)、Vespa velutina nigrithorax prepromelittin mRNA(登录号AF487908)、Vespa magnifica prepromelittin mRNA(登录号AF487910)。 利用GST基因融合表达系统构建了中华蜜蜂、亚非马蜂溶血肽基因的大肠杆菌诱导表达载体pGEX-AccM和pGEX-PhM，并在菌株BL21中进行了高效表达。二者的表达产物经Western blotting检测均具有很强的免疫活性，与预期目的蛋白分子量大小一致，三抗夹心ELISA检测结果也表明表达产物具有明显的免疫学活性。表达条件的优化实验表明pGEX-PhM和pGEX-AccM在IPTG诱导下都能够在大肠杆菌 BInl中稳定高效表达融合蛋白，两者的诱导表达对 OD。、IPTG浓度、温度以及诱导时间都有一定的选择性。在各自最佳条件而ODW为1．0时，GSTACCM表达量可以达到细菌总蛋白的12．7－14．1％，GSTPhM表达量可以达到细菌总蛋白的11．7％。溶血肽诱导家兔血小板的聚集实验也初步表明，本研究表达产物经酶解回收后得到的溶血肽蛋白是具有一定活力的，但具体活性仍有待进一步的研究。 同时，分别构建了中华蜜蜂和亚非马蜂溶血肽基因与谷肤甘肽基因融合的重组杆状病毒BacmidGSTACCM和Bacmid－GSThhM，在粉纹夜蛾细胞系Tn－SBI叶中进行了成功的表达。SDS－PAGE分析表明，感染Bacmid－GS和BaCInd七SThhM Tn细胞的表达产物均在分子量约为34kD处出现特异性条带，二者的表达量分别占Tn细胞总蛋白的7．3％和3．2％。Western blotting和三抗夹心ELISA检测同时证明二者的表达产物都具有良好的免疫活性。感染Bacmid－GSTACCM和Bacrnid－GSThhM的Tn细胞与感染阴性对照Bacmid的Tn细胞状态相比，其感染症状没有明显区别，说明表达产物融合蛋白对细胞是没有明显的毒性。溶血肽诱导家兔血小板的聚集实验也初步表明，本研究表达产物经酶解回收后得到的溶血肽蛋白是具有一定活力的，但相比之下，亚非马蜂溶血肽蛋白诱导家兔血小板的聚集活力相对较低，这与两者一级结构不同是不是有关，有待研究。 此外，我们还将中华蜜蜂和亚非马蜂溶血肽基因与增强型荧光（GppT）基因融合在杆状病毒．昆虫细胞表达系统中进行了表达，＝者的表达量分别占n细胞总蛋白的 3．1％和 2．6％。Western blotting显不表达产物对His－Tagh MonoclonalAntibody和兔抗溶血肽（与卵清蛋白交联的总蛋白）多克隆抗体都具有良好的免疫活性，在约34．6kD处均有一明显的条带，与预期目的蛋白的分子量大小一致。感染 Bacmid－GFPTAccM和 Bacmid．GFP＊hM的 Tn细胞与感染阴性对照 Bacmid的Tn细胞状态相比，其感染症状并没有明显区别，说明表达产物－融合蛋白GFPTACcM和GFPTPhM对细胞也不具明显的毒性。 同时我们也分别构建了中华蜜蜂、亚非马蜂和额斑黄胡蜂前溶血肽原基因与增强型荧光（GFPT）基因和 His Tag＋hV recognition site融合的重组杆状病毒，分别在粉纹夜蛾细胞系Tn－SBI－4中进行了表达。ELISA检测表明三者的胞内表达产物都具有良好的免疫活性，而胞外表达产物则均显阴性，说明各蜂前溶血 互互互肽原基因的连有 HIS Tag或 EGFP的前溶血肽原蛋白没有穿膜，表达产物也不能分泌出细胞外。前溶血肽原?更多还原

【Abstract】 Melittin, a peptide constituting about 40%~50% of dry venom, is the main toxin of Apis mellifera venom. It has important biological activities. It causes the lysis of erythrocytes and the release of marker ions from liposomes and of histamine from mast cells. Its amphophilic structure-a large apolar portion and a very basic polar end-is ideal for promoting interactions of melittin with lipid membranes. Extensive studies on melittin have been made in recent years and more and more attentions have been paid to its basic and clinical iatrology, biological engineering, and plant protection in agriculture.The main results in this study are shown as following:Six cDNA fragments encoding prepromelittin were amplified by RT-PCR from the total RNA of venom gland of female Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax, Vespa magnified and of worker honey bees, Apis cerana cerana and A. mellifera, respectively. The PCR products were ligated into pGEM -T easy vector. The sequencing results showed that the amplified cDNAs containing the open reading frames of prepromelittin, and their lengths were all 213 bp. The ORFs were potential to encode polypeptides of 70 amino acid residues with predicted molecular weight of 7.7 kDa, including a signal peptide of 21 residues and a promelittin of 49 residues. Comparative analysis showed that the prepromelittins from 4 Vespoidea species and 2 honeybee species shared more than 92% identities in amino acid sequences with each other. The sequences of prepromelittins of Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax, Vespa magnifica,Apis cerana cerana and Apis mellifera shared 93%, 100%, 97.2%, 97.2%, 97.2% and 100% identities in amino acid sequences with that of Apis cerana indica, respectively. In conclusion, the prepromelittins are very conserved in the primary structure and the insects of Vespoidea also contain the melittins in their venoms, which are very similar to that ofhoneybee, although they belong to different superfamilies. It is the first report in the world that there are me/ittin genes of Vespoidea species. The /vepromelittin mRNA sequences of Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax, Vespa magnified and Apis cerana cerana reported here were all submitted to GenBank with accession No. AF487909, AF487911, AF487908, AF487910 and AF487907, respectively.The me/ittin coding regions of A. cenara cenara (AccM) and P. hebraeus (PhM)were inserted into the GST fusion expression vector pGEX-4T-2 to form therecombinant vectors, pGEX-AccM and pGEX-PhM, respectively. The 2 recombinantvectors were transformed into the E. coli BL21 and the target fusion proteins were thenhighly expressed with the induction of IPTG The expressed protein bands of about29kD, which were consistent to the expected molecular weight of the fusion proteins,GST-AccM and GST-PhM(28.5kD), appeared on the SDS-PAGE profiles. Theexpression products were confirmed by Western blotting and triple antibody sandwichELISA. At the same time, the expression conditions of GST-AccM and GST-PhMfusion proteins for E. coli BL21 transformants were optimized, respectively, indifferent bacterial concentrations (ODeoo), IPTG concentrations, temperatureconditions and induction durations. Thin layer scanning on the SDS-PAGE profiles ofGST-AccM and GST-PhM showed that the expressed proteins accumulated up to about12.7-14.1% and 11.7% of total protein of bacterial cells under the optimizedexpression conditions, respectively. Purified and recovered recombinant me/ittins of A.cerana cerana and P. hebraeus showed bioactivity in activating rabbit platelets toaggregate, accompanying by the formation of thromboxane B2.The restriction fragments of GSTAccM and GSTPhM were inserted into the multiple cloning site of the pBacFastHTb to construct recombinant donor plasmaids, pBacHT-GSTAccM and pBacHT-GSTPhM, which were used to transposed to the target Bacmid in E. coli(DH10) by Tn7 transposition funct更多还原