2种蜜蜂和4种胡蜂溶血肽基因克隆与表达研究
|
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 Monoclonal
Antibody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?
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 of
honeybee, 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 the
recombinant vectors, pGEX-AccM and pGEX-PhM, respectively. The 2 recombinant
vectors were transformed into the E. coli BL21 and the target fusion proteins were then
highly expressed with the induction of IPTG The expressed protein bands of about
29kD, 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. The
expression products were confirmed by Western blotting and triple antibody sandwich
ELISA. At the same time, the expression conditions of GST-AccM and GST-PhM
fusion proteins for E. coli BL21 transformants were optimized, respectively, in
different bacterial concentrations (ODeoo), IPTG concentrations, temperature
conditions and induction durations. Thin layer scanning on the SDS-PAGE profiles of
GST-AccM and GST-PhM showed that the expressed proteins accumulated up to about
12.7-14.1% and 11.7% of total protein of bacterial cells under the optimized
expression conditions, respectively. Purified and recovered recombinant me/ittins of A.
cerana cerana and P. hebraeus showed bioactivity in activating rabbit platelets to
aggregate, 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
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 Monoclonal
Antibody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?
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 of
honeybee, 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 the
recombinant vectors, pGEX-AccM and pGEX-PhM, respectively. The 2 recombinant
vectors were transformed into the E. coli BL21 and the target fusion proteins were then
highly expressed with the induction of IPTG The expressed protein bands of about
29kD, 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. The
expression products were confirmed by Western blotting and triple antibody sandwich
ELISA. At the same time, the expression conditions of GST-AccM and GST-PhM
fusion proteins for E. coli BL21 transformants were optimized, respectively, in
different bacterial concentrations (ODeoo), IPTG concentrations, temperature
conditions and induction durations. Thin layer scanning on the SDS-PAGE profiles of
GST-AccM and GST-PhM showed that the expressed proteins accumulated up to about
12.7-14.1% and 11.7% of total protein of bacterial cells under the optimized
expression conditions, respectively. Purified and recovered recombinant me/ittins of A.
cerana cerana and P. hebraeus showed bioactivity in activating rabbit platelets to
aggregate, 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
引文
1．陈惠芳．蜂毒的研究进展．医药情报．1991，2：57～59．
2．陈盛禄．中国蜜蜂学．中国农业出版社．2001：1～777．
3．陈仲兵，张青文，李继周．蜂毒溶血肽的克隆及序列分析．农业生物技术学报．1998，6(1)：95～100．
4．丁志贤．蜂毒的研究与临床应用进展．蜜蜂杂志．2001，10：7～8．
5．杜桃柱．养蜂与蜂产品开发．山西科学技术出版社．1998：1～280．
6．房柱，杨毓鳞，周邦训．蜜蜂毒的研究与利用，见陈远聪主编，毒素的研究和利用．北京：科学出版社．1988：128～144．
7．房柱．蜂疗概论．蜜蜂杂志．1991，4：20～21．
8．葛春华，郑祖祥，周威君，孙鸿才．实用商品资源昆虫．中国农业出版社．1995：1～284．
9．葛凤晨．从蜜蜂毒中分离磷脂酶A2．蜜蜂杂志．1989，(6)：3～4．
10．古朝昆，梁秀娟．蜂毒的医疗应用与毒性反应处理．右江民族医学院学报．1996(3)：428～430．
11．韩学海，隋森芳．低浓度蜂毒素在溶液中的自聚集．生物化学与生物物理学报．1996，28(3)：307～311．
12．金冬雁，黎孟枫译，J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著．分子克隆实验指南第二版．科学出版社．1999：1～1039．
13．柯礼道．昆虫和毒素的生物化学．见陈远聪主编，毒素的研究和利用．北京：科学出版社，1988：164～182．
14．来志华译．现代的蜂毒过敏症．蜜蜂杂志．1989，1：38～39．
15．黎遵列，韩运筹．蜂毒的药理研究进展．中国中医急症．2001，10(3)：163～164．
16．李大力，王关林，方宏筠．蜂毒肽前体蛋白cDNA的克隆及序列分析，大连理工大学学报．2000，40(2)：172～175．
17．李继周，陈仲兵，蔡青年，张青文，郭忠，张福林，张芩．蜂毒溶血肽(melittin)的cDNA克隆．农业生物技术学报．1997，5(2)：162～167．
18．李绍祥，李琦，凌昌全．蜂毒素的研究新进展．中草药．2001，32(10)：942～945．
19．李铁生．中国胡蜂资源的开发与利用．科学出版社．1993：1～170．
20．李铁生．中国经济昆虫志(第三十册)膜翅目胡蜂总科．科技出版社．1985：1～159．
21．李燕红，刘飞鹏，朱嘉明，周天鸿，李月琴．抗菌肽A-蜂毒素杂合肽基因在大肠杆菌中的克隆与初步分泌表达．暨南大学学报．1999，20(5)：88～91．
22．李英华，胡福良，刘艳荷．蜂毒过敏原磷脂酶A2．生命的化学．2001，21(4)：299～302．
23．廖晓青，吴珍红，李江红，陈琪．意蜂蜂毒中游离氨基酸的分析．中国养蜂．2000，51(3)：5～6．
24．凌昌全，黄雪强，刘岭，王勇姿，张亚妮．蜂毒素缓释制剂瘤内注射减毒增效作用的实验研究．第二军医大学学报．2001，22(7)：615～617．
25．刘岭，凌昌全，胡晋红，黄雪强．蜜蜂毒中蜂毒素的分离纯化方法及含量测定．第二军医大学学报．2001，22(7)：609～411．
26．刘艳荷，陈盛禄，张传溪．蜂毒溶血肽的研究进展．昆虫知识．2001，38(6)：410～413．
27．骆尚骅．蜂毒与蜂毒过敏(综述)．中国养蜂．2001，52(5)：29～30．
28．缪晓青，吴珍红，李江红，陈琪．意蜂蜂毒中游离氨基酸的分析．中国养蜂．2000，51(3)：5～4．
29．钱锐．从蜜蜂毒中分离磷脂酶丸蜜蜂杂志．1989，6：3～4．
30．沈关心，龚非力等译，E．哈洛，D．莱恩编著．抗体技术实验指南．科学出版社．1988：1～300．
31．沈立荣，张传溪，程家安．意大利蜜蜂蜂毒透明质酸酶基因的克隆及序列分析．浙江大学-农业与生命科学版．2002，3(3)：289～292．
32．沈立荣，张传溪，程家安．中华蜜蜂、意大利蜜蜂蜂毒磷脂酶A_2：基因的克隆及序列分析．农业生物技术学报．2002，10(1)：29～32．
33．沈立荣．中蜂、意蜂蜂毒磷脂酶A2和透明质酸酶基因的克隆与表达．浙江大学博士学位论文．2002．
34．宋心仿，闫继耀，邵有全．蜜蜂产品的应用与检测加工技术．中国农业出版社．2000：1～389．
35．孙丽萍，董捷，闫继红，王凤忠，吴粹文．蜂毒肽的结构、功能及分子生物学研究进展．蜜蜂杂志．2002，8：7～9．
36．孙欣，陈维辛，陈郴永，营佳．蜂毒的医药应用研究．生化药物杂志．1990，3：15～18．
37．王凤忠，李桂芬．WTO与中国蜂产品行业．农牧产品开发．2000，(3)：6～6．
38．王关林，李大力，方宏筠．蜂毒溶血肽基因的定点诱变及其在大肠杆菌中的表达．遗传学报．2000，27(2)：176～182．
39．王华夫．我国利用蜂针、蜂毒治病起源的探讨．中国养蜂．1996，3：19～20．
40．王俊霞，郭志玲，陈红坤，王俊晓．胡蜂毒素致急性肾功能衰竭18例临床分析．现代诊断与治疗．1999，10(5)：303．
41．王秋波，吴虹，鲁迎年，张艳丽．蜂毒肽表达克隆的初步构建．青岛大学医学院学报．2001，37(1)：36～37．
42．卫应，杨申，江明华．蜂毒的药理研究、临床应用及开发现状．中国医院药学杂志．2000，20(1)：682～683．
43．肖廷自．蜂疗的历史、现状和未来．中国养蜂．2001，52(1)：19．
44．肖锡红．蜂毒的主要成份及其功能．养蜂科技．1991，3：25．
45．徐杰，陈莉莉，潘芝桂．蜂毒疗法研究概况．蜜蜂杂志．1997，11：3～5．
46．叶文．胡蜂在农林业中的人工利用及蜂毒制取．三百六十行．1995，1：32～34．
47．叶振生，许少玉，刁青云．蜂国奥秘．中国农业大学出版社．2001：1～112．
48．伊琳娜·塔图．蜂毒的抗菌作用．养蜂科技．1990，1：28～29．
49．余金良，何俊华．蜜蜂毒主要过敏原磷脂酶A2研究概述．昆虫知识．1999，36(4)：248～249．
50．余林生．蜂毒疗法的理论和实践．安徽农业技术师范学院学报．1995，9(3)：59～63．
51．余林生．国内外蜂毒的研究及其利用．养蜂科技．1995，5：24～26．
52．余林生．影响蜂毒采集的因素．蜜蜂杂志．1995，12：7～9．
53．余晓东，熊郁良．意大利蜜蜂毒 Melittin 组分的研究(Ⅱ)-活化家兔血小板聚集作用机制．重庆师范学院学报．1996，13(1)：15～20．
54．余晓东，熊郁良．意大利蜜蜂毒 Melittin 组分的研究(Ⅲ)-活化家兔血小板的释放反应．重庆师范学院学报．1996，13(1)：85～87．
55．岳玉桃，李正斌，武玉珍，年桂芝．胡蜂蜇伤后多脏器功能衰竭四例分析．洛阳医专学报．1995，14(2)：94～95．
56．张波，唐立尧．蜂毒的药理作用及应用．中国疗养医学．2001，10(4)：22～23．
57．张传溪，施惠娟，陈哲宇．EGFP基因在粉纹夜蛾细胞中的高效表达．动物学研究，1999，20(6)：468～470．
58．张传溪，吴祥甫．SA脂质体介导DNA转染昆虫细胞的研究．生物工程学报．1998，4(1)：106～108．
59．张景艳，傅小锁．肥大细胞脱颗粒肽的研究进展．国外医学药学分册．1995，22(3)：162～165．
60．张启明，李铁生，孙铁民，梁伟．胡蜂毒对肿瘤杀伤作用和治疗作用的初步报告．实用肿
瘤学杂志．1997，11(4)：260～261．
61．张素霞，杨祖洁．蜂毒中毒2例．实用儿科临床杂志．2002，17(5)：436．
62．周昌娥，刘玉，陈少秀．蜂毒致多器官功能衰竭2例．护理研究．2002，1：58～59．
63．周淑平，李英．蜜蜂溶血肽(melittin)的研究进展．生物化学与生物物理进展．1988，4：31～36．
64．朱伟，王本祥，朱迅．蜂毒素的研究进展．白求恩医科大学学报．2001，27(2)：212～214．
65. Allalouf D, Ber A, Ishay J. Hyaluronidase activity of extracts of venom sacs of a number of Vespinae (Hymenoptera). Comp. Biochem. Physiol. B. 1972, 43:119～121.
66. Allalouf D, Ber A, Ishay J. Properties of testicular hyaluronidase of th honey bee and oriental hornet: Comparison with insect venom and mammalian hyaluronidases. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 1975, 50(2): 331～337.
67. Anderson D, Terwilliger T C, Wickner W, Eisenberg D. Melittin forms crystals which are suitable for high resolution X-ray structural analysis and which reveal a molecular 2-fold axis of symmetry. The Journal of Biological Chemistry. 1980, 255(6): 2578～2582.
68. Anthony T T. Venoms: chemistry and molecular biology. A Wiley-Inter sci Publ. John Wiley & Sons, New York, USA, 1977: 501～530.
69. Barr-Nea L, Ishay J. Effect of the venom sac content of the oriental hornet (Vespa orientalis ) on the metamorphosis of the toad tadpole(Bufo viridis). Experientia. 1975, 31:212～215.
70. Christensen B, Fink J, Merrifield R B, Mauzerall D. Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc. Natl. Acad. Sci. USA. 1988, 85: 5072～5076.
71. Edery H, Ishay J, Lass L, Gitter S. Pharmacological activity of oriental hornet (Vespa orientalis) venom. Toxicon. 1972, 10: 13～19.
72. Fischl J, Ishay J, Goldberg S, GitterS. Investigation of protein fractions and haemolytic properties of wasp venom. Acta Pharmacol. Toxicol. 1972, 31: 65～70.
73. Gauldie J, Hanson J M, Rumjanek F D, Shipolini R A, Vernon C A. The peptide component of bee venom. Eur. J. Biochem. 1976, 61: 369～376.
74. Gmachl M, Kreil G. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc. Natl. Acad. Sci. USA. 1993, 90: 3569～3573.
75. Gmachl M, Kreil G. The precursors of the bee venom constituents apamin and MCD peptide are encoded by two genes in tandem which share the same 3'-exon. J. Biol. Chem. 1995, 270: 12704～12708.
76. Gunter S M D, Birger K M D, Christina R M D, Werner A M D. Rush hymenoptera venom immunotherapy: a safe and practical protocol for high-risk patients. Journal of Allergy and Clinical Immunology. 2002, 110(6): 928～933.
77. Habermann E. Bee and wasp venoms. Science. 1972, 177: 314～322.
78. Hadjipetrou-Kourounakis L, Yiangou M. Bee venom adjuvant induced disease and interleukin production. J. Rheumatol. 1988, 15(7): 1126～1128.
79. Hagler J R, Narando S E, Bradley-Dunlop D et al. A monoclonal antibody to pink bollworm(Lepidoptera: Gelechiidae)egg antigen: a tool for predator gut analysis. Ann. Entomol. Soc. Am. 1994, 87(1): 85～90.
80. Haim B, Rimon A, Ishay J S, Rimon S. Purification, characterization and anticoagulant activity of a proteolytic enzyme from Vespa orientalis venom. Toxicon. 1999, 37: 825～829.
81. Han H J, Lee J H, Park S H, Choi H J, Yang I S, Mar W C, Kang S K, Lee H J. Effect of bee venom and its melittin on apical transporters of renal proximal tubule cells. Kidney&Blood Pressure Research. 2000, 23(6): 393～399.
82. Hausley, M Z, miglierini G L, Soldatova L et al. Crystal structure of hyaluronodase, a allergen of bee venom. Sturcture. 2000, 8: 1025～1035.
83. Ho C L, Ko J L. Purification and characterization of a lethal protein with phospholipase Al activity from the hornet (Vespa basalis) venom. Biochimica et Biophysica Acta(BBA)-Lipids and Lipid Metabolism. 1988, 963(3): 414～422.
84. Ho C L, Lin Y L, Chen W C, Rocchi R, Piek T. Comparison of the immunogenicity of wasp venom peptides with or without carbohydrate moieties. Toxicon. 1998, 36(1): 217～221.
85. Ho C L, Lin Y L, Li S F. Three toxins with phospholipase activity isolated from the yellow-legged hornet (Vespa verutina) venom. Toxicon. 1999, 37:1015～1024.
86. Ho L C, Lin Y L, Chen C W, Hwang L L, Yu M H, Wang T K. Structural requirements for the dema-inducing and hemolytic activities of mastoparan B isolated from the hornet (Vespa basalis) venom. Toxicon. 1996, 34(9): 1027～1035.
87. Ishay J, Nadler E Z, Gitter S. Pharmacological activity of Paravespura germanica wasp venom.
Acta Pharmacol. Toxicol. 1973, 33: 157～160.
88. Joshua H, Ishay J. The anti-coagulant properties of an extract from the venom sac of the oriental hornet. Toxicon. 1975, 13:11～18.
89. Justin O S, Soichi Y, Makoto M, Christopher K S. Hornet venoms: lethalities and lethal capacities. Toxicon. 1986, 24(9): 950～954.
90. Kaplinsky E, Ishay J, Gitter S. Oriental hornet venom: effects on cardiovascular dynamics. Toxicon. 1974, 12: 69～73.
91. Kasuhiro K, Akiko M, Hiroaki T, Miki H, Yasuhiro I, Hideo N, Tadashi Y, Nobufumi K. α-Pompilidotoxin (α-PMTX), a novel neurotoxin from the venom of a solitary wasp, facilitates transmission in the crustacean neuromuscular synapse. Neuroscience Letters. 1997, 238: 99～102.
92. Katsuhiro K, Mario S P, Izaura Y H, Maria A J, Luiz J, Tadashi Y. Identification of bradykinins in solitary wasp venoms. Toxicon. 2002, 40: 309～312.
93. Katsuhiro K, Miki H, Hideo N, Yasuhiro I, Nobufumi K, Akiko M, Tadashi Y, Yukiko M, Yoshihiro N.. Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado). Toxicon. 2000, 38:1505～1515.
94. Katsuhiro K, Miki H, Renato F, Carla C B L, Hideo N, Yasuhiro I, Akiko M, Nobufumi K, Yoshihiro N, Tadashi Y, Joao R N, Walter F de A Jr, Mario S P, Terumi N. Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis. Biochimica et Biophysica Acta. 2001, 1550: 70～80.
95. Kemeny D M, Dalton N, Lawrence A J. et al. The purification and characterization of hyaluronidase from venom of the honey bee, Apis mellifera. Eur. J. Biochem. 1984, 139: 217～223.
96. King T P, Kochoumian L, Lam T. Immunochemical observations of antigen 5, a major venom allergen of hornets, yellowjackets and wasps. Molecular Immunology. 1987, 24(8): 857～864.
97. King T P. Immunochemical studies of stinging insect venom allergens. Toxicon. 1996, 34(11): 1455～1458.
98. Kubo H, Loegering D H, Adolphson C R. Cytotoxicproperties of eosinophil granule major basic protein for tumor cells. Int. Arch. Allergy Immunol. 1999, 118(2-4): 426～428.
99. Kuchler K, Gmachl M, Sippl M J. Analysis of the cDNA for phospholipase A2 from honeybee venom glands. The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes. Eur. J. Biochem. 1989, 184: 249～254.
100. Lariviere W R, Melzack R. The bee venom test: a new tonic-pain test. Pain. 1996, 66: 271～277.
101. Lazarev V N, Parfenova T M, Gularyan S K, Misyurina O Yu., Akopian T A, Govorun V M. Effect of controlled expression of the melittin gene on infection caused by Mycoplasma hominis in cell culture. Biochemistry, Biophysics, and Molecular Biology. 2001, 378:186～187.
102. Liu P, Davis P, Liu H, Krishnan T R. Evaluation of cytotoxicity and absorption enhancing effects of melittin-a novel absorption enhancer. European Journal of Pharmaceutics and Biopharmaceutics. 1999, 48: 85～87.
103. Maleszka R. Molecules to behaviour in the honeybee the emergence of comparative neurogenomics. Tins. 2000, 23(11): 513～514.
104. Matsuzaki K, Yoneyama S, Miyajima K. Pore formation and translocation of melittin. Biophysical Journal. 1997, 73(2): 831～838.
105. Mulfinger L, Yunginger J, Styer W et al.. Sting morphology and frequency of sting autotomy among medivally important vespidae (Hymenoptera, Vespidae) and the honey bee (Hymenoptera, Apidae). J. Medical Entomo. 1992, 29: 325～328.
106. Muller U R. Insect sting Allergy. Stuttgart and New York: Gustav Fischer. 1990.
107. Neumann W, Habermann E. Ber die phospholipase A des biennngiftes. Z. Physilo. Chem. 1954, 296: 166～169.
108. Nobufumi K, Takashi A, Shinichiro H, Akiko N. Effects of a neurotoxin in hornet venom on neuromuscular junctions of lobster. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology. 1980, 65(2): 87～92.
109. O'Connor R, Erickson R. Hymenoptera antigens: an immunological comparison of venoms, venom sac extracts and whole insect extracts. Ann. Allergy.. 1965, 23:151～155.
110. Oliveira M R, Palma M S. Polybitoxins: a group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia Paulista). Toxicon. 1998, 36(1): 189～199.
111. Owen M D, Pfaff L A, Robert E R, Wypych J. Phospholipase A2 in venom extracts from honey bees(Apis mellifera L.) of different ages. Toxicon. 1990, 28(7): 813～820.
112. Pereira S M C, Palma C M L, Pedro E, Branco F M, Spinola A, Palma C A G. Specific immunotherapy with hymenoptera venom. Clinical and Applied Immunology Reviews. 2001, 1: 249～253.
113. Prenner C, Mach L, Glossl J et al.. The antigenicity of the carbohydrate moiety of an insect glycoprotein, honeybee (Apis mellifera) venom phospholipase A2. The role of alphal-bound N-acetyglucosamine. Biochemical Journal. 1992, 284:377～380.
114. Quistad G B, Skinner W S, Schooley D A. Venom of social Hymenoptera-Toxicity to the Lepidopteran, Manduca Sexta. Insect Biochem. 1998, 18:511～514.
115. Russo A J, Cobbs C S, Ishay J S, Calton G J, Burnett J W. Isolation fo a lethal factor from venom of Vespa orientalis(oriental hornet) by affinity chromatography using cross reactive monoclonal antibody. Toxicon. 1983, 21(1): 166～169.
116. Salikhov S, Sagdiev N Z H, Korneev A S. The structure and function of biologically active compounds from the venoms of Vespa orientalis and Segestria florentina spiders. Toxicon. 1995, 33(6): 715～720.
117. Sandbank U, Ishay J, Gitter S. Mitochondrial changes in the guinea pig muscle after envenomation with Vespa orientalis venom. Experientia. 1971, 27: 303～306.
118. Schmidt J O. Biochemistry of insect venoms. Ann. Rev. Entomol. 1982, 27: 339～368.
119. Shen Li-rong, Zhang Chuanxi, Cheng Jia-an. Cloning and sequencing of gene encoding hyaluronidase from the venom of Apis cerana cerana. Entomological Sinica. 2001, 8(4): 353～360.
120. Shiplini R A, Callewaert G L. Phospholipase A2 from bee venom. Eur. J. Biochem. 1971, 20: 459～468.
121. Sigma. Biochemical and regents, for life science research. 2001-2002: 1～2843.
122. Soldatova L, Mueller U. Superior biological activity of the recombinant bee venom allergen hyaluronidase expressed in Baculovirus-infected insect cells as compared with Escherichia coli. J. Allergy Clin. Immunol. 1998, 101: 691～697.
123. Takashi A, Masato S, Tsuyoshi F, Jiro H, Michinori A. Giant hornet (Vespa mandarinia) venomous phospholipases, the purification, characterization and inhibitory properties by biscoclaurine alkaoids. Toxicon. 2000, 38:1803～1816.
124. Takashi A, Yasuaki H, Nobufumi K, Akiko M. Comparative study of amino acid compositon
in an extract from hornet venom sacs: high content of neuroactive amino acids in Vespa. Toxicon. 1989, 27(6): 683～688.
125. Terwilliger T C, Eisenberg D. The sturcture of melittin, Ⅰ structure determination and partial refinement. The Journal of Biological Chemistry. 1982, 257(11): 6010～6015.
126. Terwilliger T C, Eisenberg D. The sturcture of melittin, Ⅱ interpretation of the structure. The Journal of Biological Chemistry. 1982, 257(11): 6016～6022.
127. Visscher P K, Vetter R S. Removing bee stings: speed matters, method doesn't. The Lancet. 1996, 348: 301～302.
128. Vlasak R, Kreil G. Nucleotide sequence of cloned cDNA coding for preprosecapin, a major product of queen-bee venom glands. Eur. J. Biochem. 1984, 145: 279～282.
129. Vlasak R, Unger-ullmann C, Kreil G, Frischauf A M. Nucleotide sequence of cloned cDNA coding for honeybee prepromelittin. Eur. J. Biochem. 1983, 135: 123～126.
130. Wachinger M, Saermark T, Erfle Ⅴ. Influence of amphipathic peptides on the HIV-1 productionin persistently in fected T lymphoma cells. FEBS Lett. 1992, 309(3): 235～241.
131. Werner G, Walter V. Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects. Cell Tissue Res. 2000, 302: 271～284.
132. Wood C L, Hoffman D R. Two-dimensional polyacrylamide gel electrophoresis of Hymenoptera venom and venom sac extracts. Toxicon. 1983, 21(2): 291～299.
133. Yoshida H, Geller R G, Pisano J J. Vespulakinins: new carbohydrate containing bradykinin derivatives. Biochemistry. 1976, 15: 61～66.
2．陈盛禄．中国蜜蜂学．中国农业出版社．2001：1～777．
3．陈仲兵，张青文，李继周．蜂毒溶血肽的克隆及序列分析．农业生物技术学报．1998，6(1)：95～100．
4．丁志贤．蜂毒的研究与临床应用进展．蜜蜂杂志．2001，10：7～8．
5．杜桃柱．养蜂与蜂产品开发．山西科学技术出版社．1998：1～280．
6．房柱，杨毓鳞，周邦训．蜜蜂毒的研究与利用，见陈远聪主编，毒素的研究和利用．北京：科学出版社．1988：128～144．
7．房柱．蜂疗概论．蜜蜂杂志．1991，4：20～21．
8．葛春华，郑祖祥，周威君，孙鸿才．实用商品资源昆虫．中国农业出版社．1995：1～284．
9．葛凤晨．从蜜蜂毒中分离磷脂酶A2．蜜蜂杂志．1989，(6)：3～4．
10．古朝昆，梁秀娟．蜂毒的医疗应用与毒性反应处理．右江民族医学院学报．1996(3)：428～430．
11．韩学海，隋森芳．低浓度蜂毒素在溶液中的自聚集．生物化学与生物物理学报．1996，28(3)：307～311．
12．金冬雁，黎孟枫译，J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著．分子克隆实验指南第二版．科学出版社．1999：1～1039．
13．柯礼道．昆虫和毒素的生物化学．见陈远聪主编，毒素的研究和利用．北京：科学出版社，1988：164～182．
14．来志华译．现代的蜂毒过敏症．蜜蜂杂志．1989，1：38～39．
15．黎遵列，韩运筹．蜂毒的药理研究进展．中国中医急症．2001，10(3)：163～164．
16．李大力，王关林，方宏筠．蜂毒肽前体蛋白cDNA的克隆及序列分析，大连理工大学学报．2000，40(2)：172～175．
17．李继周，陈仲兵，蔡青年，张青文，郭忠，张福林，张芩．蜂毒溶血肽(melittin)的cDNA克隆．农业生物技术学报．1997，5(2)：162～167．
18．李绍祥，李琦，凌昌全．蜂毒素的研究新进展．中草药．2001，32(10)：942～945．
19．李铁生．中国胡蜂资源的开发与利用．科学出版社．1993：1～170．
20．李铁生．中国经济昆虫志(第三十册)膜翅目胡蜂总科．科技出版社．1985：1～159．
21．李燕红，刘飞鹏，朱嘉明，周天鸿，李月琴．抗菌肽A-蜂毒素杂合肽基因在大肠杆菌中的克隆与初步分泌表达．暨南大学学报．1999，20(5)：88～91．
22．李英华，胡福良，刘艳荷．蜂毒过敏原磷脂酶A2．生命的化学．2001，21(4)：299～302．
23．廖晓青，吴珍红，李江红，陈琪．意蜂蜂毒中游离氨基酸的分析．中国养蜂．2000，51(3)：5～6．
24．凌昌全，黄雪强，刘岭，王勇姿，张亚妮．蜂毒素缓释制剂瘤内注射减毒增效作用的实验研究．第二军医大学学报．2001，22(7)：615～617．
25．刘岭，凌昌全，胡晋红，黄雪强．蜜蜂毒中蜂毒素的分离纯化方法及含量测定．第二军医大学学报．2001，22(7)：609～411．
26．刘艳荷，陈盛禄，张传溪．蜂毒溶血肽的研究进展．昆虫知识．2001，38(6)：410～413．
27．骆尚骅．蜂毒与蜂毒过敏(综述)．中国养蜂．2001，52(5)：29～30．
28．缪晓青，吴珍红，李江红，陈琪．意蜂蜂毒中游离氨基酸的分析．中国养蜂．2000，51(3)：5～4．
29．钱锐．从蜜蜂毒中分离磷脂酶丸蜜蜂杂志．1989，6：3～4．
30．沈关心，龚非力等译，E．哈洛，D．莱恩编著．抗体技术实验指南．科学出版社．1988：1～300．
31．沈立荣，张传溪，程家安．意大利蜜蜂蜂毒透明质酸酶基因的克隆及序列分析．浙江大学-农业与生命科学版．2002，3(3)：289～292．
32．沈立荣，张传溪，程家安．中华蜜蜂、意大利蜜蜂蜂毒磷脂酶A_2：基因的克隆及序列分析．农业生物技术学报．2002，10(1)：29～32．
33．沈立荣．中蜂、意蜂蜂毒磷脂酶A2和透明质酸酶基因的克隆与表达．浙江大学博士学位论文．2002．
34．宋心仿，闫继耀，邵有全．蜜蜂产品的应用与检测加工技术．中国农业出版社．2000：1～389．
35．孙丽萍，董捷，闫继红，王凤忠，吴粹文．蜂毒肽的结构、功能及分子生物学研究进展．蜜蜂杂志．2002，8：7～9．
36．孙欣，陈维辛，陈郴永，营佳．蜂毒的医药应用研究．生化药物杂志．1990，3：15～18．
37．王凤忠，李桂芬．WTO与中国蜂产品行业．农牧产品开发．2000，(3)：6～6．
38．王关林，李大力，方宏筠．蜂毒溶血肽基因的定点诱变及其在大肠杆菌中的表达．遗传学报．2000，27(2)：176～182．
39．王华夫．我国利用蜂针、蜂毒治病起源的探讨．中国养蜂．1996，3：19～20．
40．王俊霞，郭志玲，陈红坤，王俊晓．胡蜂毒素致急性肾功能衰竭18例临床分析．现代诊断与治疗．1999，10(5)：303．
41．王秋波，吴虹，鲁迎年，张艳丽．蜂毒肽表达克隆的初步构建．青岛大学医学院学报．2001，37(1)：36～37．
42．卫应，杨申，江明华．蜂毒的药理研究、临床应用及开发现状．中国医院药学杂志．2000，20(1)：682～683．
43．肖廷自．蜂疗的历史、现状和未来．中国养蜂．2001，52(1)：19．
44．肖锡红．蜂毒的主要成份及其功能．养蜂科技．1991，3：25．
45．徐杰，陈莉莉，潘芝桂．蜂毒疗法研究概况．蜜蜂杂志．1997，11：3～5．
46．叶文．胡蜂在农林业中的人工利用及蜂毒制取．三百六十行．1995，1：32～34．
47．叶振生，许少玉，刁青云．蜂国奥秘．中国农业大学出版社．2001：1～112．
48．伊琳娜·塔图．蜂毒的抗菌作用．养蜂科技．1990，1：28～29．
49．余金良，何俊华．蜜蜂毒主要过敏原磷脂酶A2研究概述．昆虫知识．1999，36(4)：248～249．
50．余林生．蜂毒疗法的理论和实践．安徽农业技术师范学院学报．1995，9(3)：59～63．
51．余林生．国内外蜂毒的研究及其利用．养蜂科技．1995，5：24～26．
52．余林生．影响蜂毒采集的因素．蜜蜂杂志．1995，12：7～9．
53．余晓东，熊郁良．意大利蜜蜂毒 Melittin 组分的研究(Ⅱ)-活化家兔血小板聚集作用机制．重庆师范学院学报．1996，13(1)：15～20．
54．余晓东，熊郁良．意大利蜜蜂毒 Melittin 组分的研究(Ⅲ)-活化家兔血小板的释放反应．重庆师范学院学报．1996，13(1)：85～87．
55．岳玉桃，李正斌，武玉珍，年桂芝．胡蜂蜇伤后多脏器功能衰竭四例分析．洛阳医专学报．1995，14(2)：94～95．
56．张波，唐立尧．蜂毒的药理作用及应用．中国疗养医学．2001，10(4)：22～23．
57．张传溪，施惠娟，陈哲宇．EGFP基因在粉纹夜蛾细胞中的高效表达．动物学研究，1999，20(6)：468～470．
58．张传溪，吴祥甫．SA脂质体介导DNA转染昆虫细胞的研究．生物工程学报．1998，4(1)：106～108．
59．张景艳，傅小锁．肥大细胞脱颗粒肽的研究进展．国外医学药学分册．1995，22(3)：162～165．
60．张启明，李铁生，孙铁民，梁伟．胡蜂毒对肿瘤杀伤作用和治疗作用的初步报告．实用肿
瘤学杂志．1997，11(4)：260～261．
61．张素霞，杨祖洁．蜂毒中毒2例．实用儿科临床杂志．2002，17(5)：436．
62．周昌娥，刘玉，陈少秀．蜂毒致多器官功能衰竭2例．护理研究．2002，1：58～59．
63．周淑平，李英．蜜蜂溶血肽(melittin)的研究进展．生物化学与生物物理进展．1988，4：31～36．
64．朱伟，王本祥，朱迅．蜂毒素的研究进展．白求恩医科大学学报．2001，27(2)：212～214．
65. Allalouf D, Ber A, Ishay J. Hyaluronidase activity of extracts of venom sacs of a number of Vespinae (Hymenoptera). Comp. Biochem. Physiol. B. 1972, 43:119～121.
66. Allalouf D, Ber A, Ishay J. Properties of testicular hyaluronidase of th honey bee and oriental hornet: Comparison with insect venom and mammalian hyaluronidases. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 1975, 50(2): 331～337.
67. Anderson D, Terwilliger T C, Wickner W, Eisenberg D. Melittin forms crystals which are suitable for high resolution X-ray structural analysis and which reveal a molecular 2-fold axis of symmetry. The Journal of Biological Chemistry. 1980, 255(6): 2578～2582.
68. Anthony T T. Venoms: chemistry and molecular biology. A Wiley-Inter sci Publ. John Wiley & Sons, New York, USA, 1977: 501～530.
69. Barr-Nea L, Ishay J. Effect of the venom sac content of the oriental hornet (Vespa orientalis ) on the metamorphosis of the toad tadpole(Bufo viridis). Experientia. 1975, 31:212～215.
70. Christensen B, Fink J, Merrifield R B, Mauzerall D. Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc. Natl. Acad. Sci. USA. 1988, 85: 5072～5076.
71. Edery H, Ishay J, Lass L, Gitter S. Pharmacological activity of oriental hornet (Vespa orientalis) venom. Toxicon. 1972, 10: 13～19.
72. Fischl J, Ishay J, Goldberg S, GitterS. Investigation of protein fractions and haemolytic properties of wasp venom. Acta Pharmacol. Toxicol. 1972, 31: 65～70.
73. Gauldie J, Hanson J M, Rumjanek F D, Shipolini R A, Vernon C A. The peptide component of bee venom. Eur. J. Biochem. 1976, 61: 369～376.
74. Gmachl M, Kreil G. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc. Natl. Acad. Sci. USA. 1993, 90: 3569～3573.
75. Gmachl M, Kreil G. The precursors of the bee venom constituents apamin and MCD peptide are encoded by two genes in tandem which share the same 3'-exon. J. Biol. Chem. 1995, 270: 12704～12708.
76. Gunter S M D, Birger K M D, Christina R M D, Werner A M D. Rush hymenoptera venom immunotherapy: a safe and practical protocol for high-risk patients. Journal of Allergy and Clinical Immunology. 2002, 110(6): 928～933.
77. Habermann E. Bee and wasp venoms. Science. 1972, 177: 314～322.
78. Hadjipetrou-Kourounakis L, Yiangou M. Bee venom adjuvant induced disease and interleukin production. J. Rheumatol. 1988, 15(7): 1126～1128.
79. Hagler J R, Narando S E, Bradley-Dunlop D et al. A monoclonal antibody to pink bollworm(Lepidoptera: Gelechiidae)egg antigen: a tool for predator gut analysis. Ann. Entomol. Soc. Am. 1994, 87(1): 85～90.
80. Haim B, Rimon A, Ishay J S, Rimon S. Purification, characterization and anticoagulant activity of a proteolytic enzyme from Vespa orientalis venom. Toxicon. 1999, 37: 825～829.
81. Han H J, Lee J H, Park S H, Choi H J, Yang I S, Mar W C, Kang S K, Lee H J. Effect of bee venom and its melittin on apical transporters of renal proximal tubule cells. Kidney&Blood Pressure Research. 2000, 23(6): 393～399.
82. Hausley, M Z, miglierini G L, Soldatova L et al. Crystal structure of hyaluronodase, a allergen of bee venom. Sturcture. 2000, 8: 1025～1035.
83. Ho C L, Ko J L. Purification and characterization of a lethal protein with phospholipase Al activity from the hornet (Vespa basalis) venom. Biochimica et Biophysica Acta(BBA)-Lipids and Lipid Metabolism. 1988, 963(3): 414～422.
84. Ho C L, Lin Y L, Chen W C, Rocchi R, Piek T. Comparison of the immunogenicity of wasp venom peptides with or without carbohydrate moieties. Toxicon. 1998, 36(1): 217～221.
85. Ho C L, Lin Y L, Li S F. Three toxins with phospholipase activity isolated from the yellow-legged hornet (Vespa verutina) venom. Toxicon. 1999, 37:1015～1024.
86. Ho L C, Lin Y L, Chen C W, Hwang L L, Yu M H, Wang T K. Structural requirements for the dema-inducing and hemolytic activities of mastoparan B isolated from the hornet (Vespa basalis) venom. Toxicon. 1996, 34(9): 1027～1035.
87. Ishay J, Nadler E Z, Gitter S. Pharmacological activity of Paravespura germanica wasp venom.
Acta Pharmacol. Toxicol. 1973, 33: 157～160.
88. Joshua H, Ishay J. The anti-coagulant properties of an extract from the venom sac of the oriental hornet. Toxicon. 1975, 13:11～18.
89. Justin O S, Soichi Y, Makoto M, Christopher K S. Hornet venoms: lethalities and lethal capacities. Toxicon. 1986, 24(9): 950～954.
90. Kaplinsky E, Ishay J, Gitter S. Oriental hornet venom: effects on cardiovascular dynamics. Toxicon. 1974, 12: 69～73.
91. Kasuhiro K, Akiko M, Hiroaki T, Miki H, Yasuhiro I, Hideo N, Tadashi Y, Nobufumi K. α-Pompilidotoxin (α-PMTX), a novel neurotoxin from the venom of a solitary wasp, facilitates transmission in the crustacean neuromuscular synapse. Neuroscience Letters. 1997, 238: 99～102.
92. Katsuhiro K, Mario S P, Izaura Y H, Maria A J, Luiz J, Tadashi Y. Identification of bradykinins in solitary wasp venoms. Toxicon. 2002, 40: 309～312.
93. Katsuhiro K, Miki H, Hideo N, Yasuhiro I, Nobufumi K, Akiko M, Tadashi Y, Yukiko M, Yoshihiro N.. Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado). Toxicon. 2000, 38:1505～1515.
94. Katsuhiro K, Miki H, Renato F, Carla C B L, Hideo N, Yasuhiro I, Akiko M, Nobufumi K, Yoshihiro N, Tadashi Y, Joao R N, Walter F de A Jr, Mario S P, Terumi N. Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis. Biochimica et Biophysica Acta. 2001, 1550: 70～80.
95. Kemeny D M, Dalton N, Lawrence A J. et al. The purification and characterization of hyaluronidase from venom of the honey bee, Apis mellifera. Eur. J. Biochem. 1984, 139: 217～223.
96. King T P, Kochoumian L, Lam T. Immunochemical observations of antigen 5, a major venom allergen of hornets, yellowjackets and wasps. Molecular Immunology. 1987, 24(8): 857～864.
97. King T P. Immunochemical studies of stinging insect venom allergens. Toxicon. 1996, 34(11): 1455～1458.
98. Kubo H, Loegering D H, Adolphson C R. Cytotoxicproperties of eosinophil granule major basic protein for tumor cells. Int. Arch. Allergy Immunol. 1999, 118(2-4): 426～428.
99. Kuchler K, Gmachl M, Sippl M J. Analysis of the cDNA for phospholipase A2 from honeybee venom glands. The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes. Eur. J. Biochem. 1989, 184: 249～254.
100. Lariviere W R, Melzack R. The bee venom test: a new tonic-pain test. Pain. 1996, 66: 271～277.
101. Lazarev V N, Parfenova T M, Gularyan S K, Misyurina O Yu., Akopian T A, Govorun V M. Effect of controlled expression of the melittin gene on infection caused by Mycoplasma hominis in cell culture. Biochemistry, Biophysics, and Molecular Biology. 2001, 378:186～187.
102. Liu P, Davis P, Liu H, Krishnan T R. Evaluation of cytotoxicity and absorption enhancing effects of melittin-a novel absorption enhancer. European Journal of Pharmaceutics and Biopharmaceutics. 1999, 48: 85～87.
103. Maleszka R. Molecules to behaviour in the honeybee the emergence of comparative neurogenomics. Tins. 2000, 23(11): 513～514.
104. Matsuzaki K, Yoneyama S, Miyajima K. Pore formation and translocation of melittin. Biophysical Journal. 1997, 73(2): 831～838.
105. Mulfinger L, Yunginger J, Styer W et al.. Sting morphology and frequency of sting autotomy among medivally important vespidae (Hymenoptera, Vespidae) and the honey bee (Hymenoptera, Apidae). J. Medical Entomo. 1992, 29: 325～328.
106. Muller U R. Insect sting Allergy. Stuttgart and New York: Gustav Fischer. 1990.
107. Neumann W, Habermann E. Ber die phospholipase A des biennngiftes. Z. Physilo. Chem. 1954, 296: 166～169.
108. Nobufumi K, Takashi A, Shinichiro H, Akiko N. Effects of a neurotoxin in hornet venom on neuromuscular junctions of lobster. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology. 1980, 65(2): 87～92.
109. O'Connor R, Erickson R. Hymenoptera antigens: an immunological comparison of venoms, venom sac extracts and whole insect extracts. Ann. Allergy.. 1965, 23:151～155.
110. Oliveira M R, Palma M S. Polybitoxins: a group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia Paulista). Toxicon. 1998, 36(1): 189～199.
111. Owen M D, Pfaff L A, Robert E R, Wypych J. Phospholipase A2 in venom extracts from honey bees(Apis mellifera L.) of different ages. Toxicon. 1990, 28(7): 813～820.
112. Pereira S M C, Palma C M L, Pedro E, Branco F M, Spinola A, Palma C A G. Specific immunotherapy with hymenoptera venom. Clinical and Applied Immunology Reviews. 2001, 1: 249～253.
113. Prenner C, Mach L, Glossl J et al.. The antigenicity of the carbohydrate moiety of an insect glycoprotein, honeybee (Apis mellifera) venom phospholipase A2. The role of alphal-bound N-acetyglucosamine. Biochemical Journal. 1992, 284:377～380.
114. Quistad G B, Skinner W S, Schooley D A. Venom of social Hymenoptera-Toxicity to the Lepidopteran, Manduca Sexta. Insect Biochem. 1998, 18:511～514.
115. Russo A J, Cobbs C S, Ishay J S, Calton G J, Burnett J W. Isolation fo a lethal factor from venom of Vespa orientalis(oriental hornet) by affinity chromatography using cross reactive monoclonal antibody. Toxicon. 1983, 21(1): 166～169.
116. Salikhov S, Sagdiev N Z H, Korneev A S. The structure and function of biologically active compounds from the venoms of Vespa orientalis and Segestria florentina spiders. Toxicon. 1995, 33(6): 715～720.
117. Sandbank U, Ishay J, Gitter S. Mitochondrial changes in the guinea pig muscle after envenomation with Vespa orientalis venom. Experientia. 1971, 27: 303～306.
118. Schmidt J O. Biochemistry of insect venoms. Ann. Rev. Entomol. 1982, 27: 339～368.
119. Shen Li-rong, Zhang Chuanxi, Cheng Jia-an. Cloning and sequencing of gene encoding hyaluronidase from the venom of Apis cerana cerana. Entomological Sinica. 2001, 8(4): 353～360.
120. Shiplini R A, Callewaert G L. Phospholipase A2 from bee venom. Eur. J. Biochem. 1971, 20: 459～468.
121. Sigma. Biochemical and regents, for life science research. 2001-2002: 1～2843.
122. Soldatova L, Mueller U. Superior biological activity of the recombinant bee venom allergen hyaluronidase expressed in Baculovirus-infected insect cells as compared with Escherichia coli. J. Allergy Clin. Immunol. 1998, 101: 691～697.
123. Takashi A, Masato S, Tsuyoshi F, Jiro H, Michinori A. Giant hornet (Vespa mandarinia) venomous phospholipases, the purification, characterization and inhibitory properties by biscoclaurine alkaoids. Toxicon. 2000, 38:1803～1816.
124. Takashi A, Yasuaki H, Nobufumi K, Akiko M. Comparative study of amino acid compositon
in an extract from hornet venom sacs: high content of neuroactive amino acids in Vespa. Toxicon. 1989, 27(6): 683～688.
125. Terwilliger T C, Eisenberg D. The sturcture of melittin, Ⅰ structure determination and partial refinement. The Journal of Biological Chemistry. 1982, 257(11): 6010～6015.
126. Terwilliger T C, Eisenberg D. The sturcture of melittin, Ⅱ interpretation of the structure. The Journal of Biological Chemistry. 1982, 257(11): 6016～6022.
127. Visscher P K, Vetter R S. Removing bee stings: speed matters, method doesn't. The Lancet. 1996, 348: 301～302.
128. Vlasak R, Kreil G. Nucleotide sequence of cloned cDNA coding for preprosecapin, a major product of queen-bee venom glands. Eur. J. Biochem. 1984, 145: 279～282.
129. Vlasak R, Unger-ullmann C, Kreil G, Frischauf A M. Nucleotide sequence of cloned cDNA coding for honeybee prepromelittin. Eur. J. Biochem. 1983, 135: 123～126.
130. Wachinger M, Saermark T, Erfle Ⅴ. Influence of amphipathic peptides on the HIV-1 productionin persistently in fected T lymphoma cells. FEBS Lett. 1992, 309(3): 235～241.
131. Werner G, Walter V. Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects. Cell Tissue Res. 2000, 302: 271～284.
132. Wood C L, Hoffman D R. Two-dimensional polyacrylamide gel electrophoresis of Hymenoptera venom and venom sac extracts. Toxicon. 1983, 21(2): 291～299.
133. Yoshida H, Geller R G, Pisano J J. Vespulakinins: new carbohydrate containing bradykinin derivatives. Biochemistry. 1976, 15: 61～66.