棉铃虫单粒包埋型核型多角体病毒p40、chi基因克隆及chi基因在大肠杆菌和昆虫细胞中的表达
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摘要
本文通过建立了棉铃虫（Helicoverpa armigera）单粒包埋型核型多角体病毒（HaSNPV）
Cl株基因组文库，并对一些克隆进行限制性内切酶分析、Southern杂交和序列测定鉴别出
了编码p40基因全序列的HindIII-H、J片段及编码几丁质酶基因的HindIII-E片段，进
一步分析了p40基因序列和几丁质酶基因序列，并在大肠杆菌和昆虫细胞中表达了几丁质
酶基因。
在棉铃虫虫体内增殖了HaSNPV Cl株，从提纯多角体中提取基因组DNA。应用BamHI、
EcoRI、EcoRV、HindIII、PstI、XbaI和XhoI 7种限制性内切酶分别将基因组DNA完全酶
切，条带数分别为11、19、22、13、6、16和5。以λDNA/HindIII酶切片段作为分子量标
准，求得各片段的长度，推测出HaSNPV基因组大小为130kb左右，进一步选用HindIII、
XbaI、XhoI、HindIII+XbaI、HindIII+PstI、HindIII+XhoI完全酶切基因组DNA，构建了
HaSNPV基因组文库。
用BamHI、EcoRI、HindIII、PstI四种限制性内切酶分析基因组文库中的克隆，筛选
出了基因组HindIII-E、H和J片段，其长度分别为10.6、10和6.7kb。对H和J片段末
端测序得到HaSNPV p40基因全序列，HindIII-H和J片段分别编码其上游和下游序列。HaSNPV
p40基因编码区全长966bp，与HzSNPV p40基因有98％的相同性，其翻译起始位点上游都
有一个保守的晚期转录起始位点ATAAG。从HaSNPV p40基因核苷酸序列推测其编码蛋白分
子量为36.58kD，其氨基酸序列与BmNPV p40和AcMNPV gp41的氨基酸序列相同性为55％
左右，但HaSNPV p40的28-277氨基酸区域与SeMNPV、LdMNPV、AcMNPV、BmNPV、OpMNPV
的同源基因相对应区域比较，相似性在60％以上，并且七种基因中存在3个保守的半胱氨酸。
对以上七种同源基因编码的氨基酸序列分析，结果表明P40蛋白溶解性都低于30％。进一步
绘出了七种杆状病毒P40蛋白进化树。
在对HaSNPV基因组HindIII-E片段限制性内切酶分析基础上，通过建立该片段随机
克隆，测定了E片段全序列。该片段含有完整的、以ATG为起始密码子、编码多肽不少于
50个氨基酸的ORF 9个，其中包括编码几丁质酶基因的ORF。HaSNPV几丁质酶基因编码区
全长1713bp，推测编码蛋白分子量为65.5kD，与HzSNPV几丁质酶基因编码的氨基酸序列
有91％的相同性，与BmNPV和AcMNPV几丁质酶基因编码的氨基酸序列相同性为66％，与粘
质沙雷氏菌（Serratia marcescens）几丁质酶A（chiA）基因编码的氨基酸序列相同性为
55％。HaSNPV几丁质酶对应于S. marcescens chiA的催化区域有73％相同性、86％相似性。
HaSNPV几丁质酶对应于S. marcescens chiA纤粘连蛋白III型组件结构区域相同性达41％，
相似性为58％。通过与HzNPV几丁质酶基因编码氨基酸序列比较识别出其N端有一个由20
个疏水性氨基酸组成的假定信号肽序列，C端有一个假定的内质网保留信号序列HNEL。
根据HaNPV几丁质酶基因5’端编码信号肽序列的假定，从基因组HindIII-E片段中
扩增了HaSNPV几丁质酶全部编码区基因（chi）和HaNPV缺失假定信号肽的几丁质酶的编码
区基因（chjs人习Chf。Chjs书其阅读框构建到pErl’28a载体中，得到pETchi、pETcllis－
两个表达克隆。pETchi、pEI’chis－转入大肠杆菌皿3中诱导表达，通过SDS－PAGE及Western
印迹检测，确定表达出分子量为60kD人右的蛋臼。
将PCR扩增出的HaSN＊ chls－基因构建到杆状病毒－昆虫表达系统的供体质粒
pFASTBACHTa中，通过转座作用将目的基因重组进穿梭载体Bacmid中，重组Bacmid在
LIPfectin介导下共转染粉纹夜蛾细胞．连续传代感染四次后，PCR检测到细胞中含重组有
目的基因的病毒，对感染细胞进行SDS干AGE分析，表明有特异性表达条带，其分子量为60M
左右。
本论文的创新点主要有：
1．首次克隆分析了flaSN。V vio基因，井确定了11aSNPV基因组 ／llndlll－11． J片段的
精确物理图谱。
2．克隆了包含几丁质酶基因的 IlaSNPV基因组 Ijlndlll｛片段，测定了 E片段全序列。
3．在大肠杆菌中高效表达了HaSNPV儿丁“质酶基因及缺失假定信号肽的几丁质酶基
因。
4．在昆虫细胞中高效表达了llaSNPV缺失假定信号肽的儿丁质酶基因。
本文尤其较系统研究了！laSNPV几丁质酶基因，为进一步的应用研究提供了基础
The genomic DNA library of plaque-purified Cl clone of Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) was constructed. Two ORFs encoding p40 and chitinase, respectively, were identified and analysed. Furthermore, expression of the chitinase gene both in E. coli and insect cell was also conducted in this paper.
HaSNPV C1 clone was propagated by infecting third instar //. armigera larvae with the viral polyhedra. Genomic DNA was extracted from purified polyhedra. Digestion of genomic DNA with 7 restriction endoriucleases( BamH I, EcoR I, EcoR V,Hind, Pst I, Xba I and Xho I) resulted in 11,19, 22, 13, 6, 16 and 5 fragments (longer than 0.8kb), respectively. Length of these fragments was calculated by comparing with A DNA/Hind III marker, and the genomic DNA length was predicted about 130 kb. Based on the restriction analysis, the genomic library was constructed by clonning the fragments produced by digestion of the genomic DNA with Hind III,Xba I, Xho I,HindIII+Xba I, HindIII+PstI, HindIII+XhoI.
The HaSNPV HindIII-H , J fragments clones encoding a Helicoverpa zea nucleopolyhedrovirus(HzNPV) p40 like gene were screened by restriction analysis and terminal sequencing methods. The HaSNPV p40 gene exhibits 98% identity with HzSNPV p40 gene at the nucleotide sequence and has a conserved baculovirus late transcription strart motif ATAAG. The HaSNPV p40 is potential to encode a protein of predicted size 36. 58 kD that has about 55% identity with BmNPV/p40 and AcMNPV gp41. Comparing HaSNPV p40 residue 28-277 region which have a hydrophilic domain with SeMNPV, LdMNPV, AcMNPV, BmNPV and OpMNPV homologue,they have above 60% similarity. Among 7 predicted protein,3 cycteines are conserved.A phylogenetic tree of 7 baculovirus p40 gene was drawn by using Genetyx programe .The result showed that the evolutionary rate of p40 genes is in some degree different to that of polyhedrin genes.
The HaSNPV HindIII-E fragment (10.6 kb) was cloned and sequenced. 9 ORFs, which are initiated with a methionine codon and encode a polypeptide of at least 50 amino acids,including the ORF coding the chitinase, were identified. The chitinase gene encoded a predicted 65.5 kD peptide which had 91%, 66% and 55%
identity with HzSNPV chitinase, AcMNPV chitinase and Serratia marcescens chiA, respectively. The putative catalytic domains of HaSNPV chitinase and Serratia marcescens chih share 78% identity and 86% similarity. A signal peptide and endoplasmic reticulum(ER) location motif were identified by comparing with HzNPV chitinase gene.
The complete chitinase gene (chi) and that deficient putative signal peptide encoding region (chis-} were amplified from HaSNPV Hindlll-E fragment by using PCR method. These two target genes were inserted into the expression vector pET28a, under the control of phage T7 promoter and with a His tag in their N terminal, respectively. Then the recombinant vectors were transformed into ?coli (DE3) for expression. An expressed band of about 60 kD was identified by SOS-poly acrylamide gel electrophoresis and further confirmed by Western blot.
chiS- gene was inserted into donor plasmid pFASTBACHTa under the control of Ph promoter and flanked by the left and right ends of Tn7. The target gene was transposed to the target Bacmid in ?coli (1)1110), by Tn7 transposition function. Recombinant Bacmid was screened by intra-allelic complementation and antibiotic resistance methods and then transfected into Tn-5Bl~4 cell mediated by Lipofectin . An expressed protein band of 60 kD was determined by SDS-PAGE.
Cl株基因组文库，并对一些克隆进行限制性内切酶分析、Southern杂交和序列测定鉴别出
了编码p40基因全序列的HindIII-H、J片段及编码几丁质酶基因的HindIII-E片段，进
一步分析了p40基因序列和几丁质酶基因序列，并在大肠杆菌和昆虫细胞中表达了几丁质
酶基因。
在棉铃虫虫体内增殖了HaSNPV Cl株，从提纯多角体中提取基因组DNA。应用BamHI、
EcoRI、EcoRV、HindIII、PstI、XbaI和XhoI 7种限制性内切酶分别将基因组DNA完全酶
切，条带数分别为11、19、22、13、6、16和5。以λDNA/HindIII酶切片段作为分子量标
准，求得各片段的长度，推测出HaSNPV基因组大小为130kb左右，进一步选用HindIII、
XbaI、XhoI、HindIII+XbaI、HindIII+PstI、HindIII+XhoI完全酶切基因组DNA，构建了
HaSNPV基因组文库。
用BamHI、EcoRI、HindIII、PstI四种限制性内切酶分析基因组文库中的克隆，筛选
出了基因组HindIII-E、H和J片段，其长度分别为10.6、10和6.7kb。对H和J片段末
端测序得到HaSNPV p40基因全序列，HindIII-H和J片段分别编码其上游和下游序列。HaSNPV
p40基因编码区全长966bp，与HzSNPV p40基因有98％的相同性，其翻译起始位点上游都
有一个保守的晚期转录起始位点ATAAG。从HaSNPV p40基因核苷酸序列推测其编码蛋白分
子量为36.58kD，其氨基酸序列与BmNPV p40和AcMNPV gp41的氨基酸序列相同性为55％
左右，但HaSNPV p40的28-277氨基酸区域与SeMNPV、LdMNPV、AcMNPV、BmNPV、OpMNPV
的同源基因相对应区域比较，相似性在60％以上，并且七种基因中存在3个保守的半胱氨酸。
对以上七种同源基因编码的氨基酸序列分析，结果表明P40蛋白溶解性都低于30％。进一步
绘出了七种杆状病毒P40蛋白进化树。
在对HaSNPV基因组HindIII-E片段限制性内切酶分析基础上，通过建立该片段随机
克隆，测定了E片段全序列。该片段含有完整的、以ATG为起始密码子、编码多肽不少于
50个氨基酸的ORF 9个，其中包括编码几丁质酶基因的ORF。HaSNPV几丁质酶基因编码区
全长1713bp，推测编码蛋白分子量为65.5kD，与HzSNPV几丁质酶基因编码的氨基酸序列
有91％的相同性，与BmNPV和AcMNPV几丁质酶基因编码的氨基酸序列相同性为66％，与粘
质沙雷氏菌（Serratia marcescens）几丁质酶A（chiA）基因编码的氨基酸序列相同性为
55％。HaSNPV几丁质酶对应于S. marcescens chiA的催化区域有73％相同性、86％相似性。
HaSNPV几丁质酶对应于S. marcescens chiA纤粘连蛋白III型组件结构区域相同性达41％，
相似性为58％。通过与HzNPV几丁质酶基因编码氨基酸序列比较识别出其N端有一个由20
个疏水性氨基酸组成的假定信号肽序列，C端有一个假定的内质网保留信号序列HNEL。
根据HaNPV几丁质酶基因5’端编码信号肽序列的假定，从基因组HindIII-E片段中
扩增了HaSNPV几丁质酶全部编码区基因（chi）和HaNPV缺失假定信号肽的几丁质酶的编码
区基因（chjs人习Chf。Chjs书其阅读框构建到pErl’28a载体中，得到pETchi、pETcllis－
两个表达克隆。pETchi、pEI’chis－转入大肠杆菌皿3中诱导表达，通过SDS－PAGE及Western
印迹检测，确定表达出分子量为60kD人右的蛋臼。
将PCR扩增出的HaSN＊ chls－基因构建到杆状病毒－昆虫表达系统的供体质粒
pFASTBACHTa中，通过转座作用将目的基因重组进穿梭载体Bacmid中，重组Bacmid在
LIPfectin介导下共转染粉纹夜蛾细胞．连续传代感染四次后，PCR检测到细胞中含重组有
目的基因的病毒，对感染细胞进行SDS干AGE分析，表明有特异性表达条带，其分子量为60M
左右。
本论文的创新点主要有：
1．首次克隆分析了flaSN。V vio基因，井确定了11aSNPV基因组 ／llndlll－11． J片段的
精确物理图谱。
2．克隆了包含几丁质酶基因的 IlaSNPV基因组 Ijlndlll｛片段，测定了 E片段全序列。
3．在大肠杆菌中高效表达了HaSNPV儿丁“质酶基因及缺失假定信号肽的几丁质酶基
因。
4．在昆虫细胞中高效表达了llaSNPV缺失假定信号肽的儿丁质酶基因。
本文尤其较系统研究了！laSNPV几丁质酶基因，为进一步的应用研究提供了基础
The genomic DNA library of plaque-purified Cl clone of Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) was constructed. Two ORFs encoding p40 and chitinase, respectively, were identified and analysed. Furthermore, expression of the chitinase gene both in E. coli and insect cell was also conducted in this paper.
HaSNPV C1 clone was propagated by infecting third instar //. armigera larvae with the viral polyhedra. Genomic DNA was extracted from purified polyhedra. Digestion of genomic DNA with 7 restriction endoriucleases( BamH I, EcoR I, EcoR V,Hind, Pst I, Xba I and Xho I) resulted in 11,19, 22, 13, 6, 16 and 5 fragments (longer than 0.8kb), respectively. Length of these fragments was calculated by comparing with A DNA/Hind III marker, and the genomic DNA length was predicted about 130 kb. Based on the restriction analysis, the genomic library was constructed by clonning the fragments produced by digestion of the genomic DNA with Hind III,Xba I, Xho I,HindIII+Xba I, HindIII+PstI, HindIII+XhoI.
The HaSNPV HindIII-H , J fragments clones encoding a Helicoverpa zea nucleopolyhedrovirus(HzNPV) p40 like gene were screened by restriction analysis and terminal sequencing methods. The HaSNPV p40 gene exhibits 98% identity with HzSNPV p40 gene at the nucleotide sequence and has a conserved baculovirus late transcription strart motif ATAAG. The HaSNPV p40 is potential to encode a protein of predicted size 36. 58 kD that has about 55% identity with BmNPV/p40 and AcMNPV gp41. Comparing HaSNPV p40 residue 28-277 region which have a hydrophilic domain with SeMNPV, LdMNPV, AcMNPV, BmNPV and OpMNPV homologue,they have above 60% similarity. Among 7 predicted protein,3 cycteines are conserved.A phylogenetic tree of 7 baculovirus p40 gene was drawn by using Genetyx programe .The result showed that the evolutionary rate of p40 genes is in some degree different to that of polyhedrin genes.
The HaSNPV HindIII-E fragment (10.6 kb) was cloned and sequenced. 9 ORFs, which are initiated with a methionine codon and encode a polypeptide of at least 50 amino acids,including the ORF coding the chitinase, were identified. The chitinase gene encoded a predicted 65.5 kD peptide which had 91%, 66% and 55%
identity with HzSNPV chitinase, AcMNPV chitinase and Serratia marcescens chiA, respectively. The putative catalytic domains of HaSNPV chitinase and Serratia marcescens chih share 78% identity and 86% similarity. A signal peptide and endoplasmic reticulum(ER) location motif were identified by comparing with HzNPV chitinase gene.
The complete chitinase gene (chi) and that deficient putative signal peptide encoding region (chis-} were amplified from HaSNPV Hindlll-E fragment by using PCR method. These two target genes were inserted into the expression vector pET28a, under the control of phage T7 promoter and with a His tag in their N terminal, respectively. Then the recombinant vectors were transformed into ?coli (DE3) for expression. An expressed band of about 60 kD was identified by SOS-poly acrylamide gel electrophoresis and further confirmed by Western blot.
chiS- gene was inserted into donor plasmid pFASTBACHTa under the control of Ph promoter and flanked by the left and right ends of Tn7. The target gene was transposed to the target Bacmid in ?coli (1)1110), by Tn7 transposition function. Recombinant Bacmid was screened by intra-allelic complementation and antibiotic resistance methods and then transfected into Tn-5Bl~4 cell mediated by Lipofectin . An expressed protein band of 60 kD was determined by SDS-PAGE.
引文
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24．张传溪等，HaSNPV 蛋白激酶基因的核苷酸序列研究，生物化学与生物物理学报，1997，29(3)：322-326
25．张传溪等，棉铃虫核型多角体病毒 HindⅢ-K 片段的核苷酸序列及其分析，中国病毒学，1998，13，(2)：142-149
26．张传溪等，EGFP 基因在粉蚊夜蛾中的高效表达，动物学研究，1999，20(6)：468-470
27．张传溪，贡成良等，杆状病毒包涵体蛋白基因的进化，浙江农业大学学报，1999，25(1)：69—76
28．张传溪，武家才．棉铃虫核型多角体病毒基因组结构及p10基因．生物化学与生物物理学报，生物化学与生物物理学报，2001，33(2)：179-184
29．钟江等，棉铃虫细胞系SFE-IIA—8212的克隆化及对HaSNPV 受纳性提高的克隆系的建立，复旦学报(自然科学版)，1994，33(2)：148-156
30．朱雪峰，陈崇顺，郁志芳．植物几丁质酶的生物功能．生命的化学，2000，20(1)：36～37
31．Bonneng B c &Hammock B D,基因工程杆状病毒杀虫剂的发展与潜力．国外农学-蚕业，1993(4)：12-21
32．J 萨姆布鲁 E F，弗里奇 T 曼尼阿蒂斯著，金冬雁，黎盂枫等译，分子克隆实验指南
[M].1993，科学出版社
33． Bhushan-Bharat; Hoondal-G-S Effect of fungicides, insecticides and allosamidin on a thermostable chitinase from Bacillus sp. BG-11． June, World-Journal-of-Microbiology-and-Biotechnology. 1999,15 (3) , 403-404．
34． Blaak H ,Schnellmann J ,Walter S , Henrissat B. Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, pupative protein domains and relationship to other chitinase. Eur J Biochem . 1993,214,659-669．
35． Chaerchomsri S,Ikeda M, Kobayashi M. Nucleotide sequence of the Lymantria dispar nuclear ployhedrosis virus DNA polymerase gene region, Journal of General Virology, 1992, 73,3177-3183．
36． Chen X W, Sun X L, HU Z M et al., Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide. Journal of invertebrate pathology, 2000, 76,140-146．
37． Chernin L. S., Fuente de la, Sobolev L. V. Molecular cloning, structural analysis, and expression in Escherichia coli. of a chitinase gene from Enterobacter agglomerans. 1997,63,834-839．
38． Cowan P, Bulach D, et al. , Nucleotide Sequence of the Polyhedrin Gene Region of Helicoverpa zea single nucleocapsid nuclear polyhedrosis virus: Placement of the Virus in Lepidopteran nuclear polygidrosis virus groupⅡ, Journal of General virology, 1994,75,3211-3218．
39． Ding X.Manduca chitinase-mediated resistance to tobacco budworm(Heliothis mirescens)and tobacco hornworm (Manduca scxta) larvae in transgenic tobacco plants. Ph. D. Dissertation, Kansas State University, Manhattan. 82 p.
40． Ding X, Gopalakrishnan B, Johnson L B, White F F, Wang X, Morgan T D, Kramer K J, et al.,Insect resistance of transgenic tabacco expressing an insert chitinase gene. Transgen. Res. 1997,75,3211-3218．
41． El-Sayed G N,Coudron T A, Ignoffo C M.Chitinolytic activity and vurulence associated with native and mutant isolates of the entomopathogenic fungus, Nomuraea ileyi.J. Invertr. Pathol., 1989,54,394-403．
42． Fraser M J, Ultrastructural observations of virion maturation in Autographa californica nuclear polyhedrosis virus infected Spodop-tera frugiperda cell cultures [J]. J. Ultrast. Mol. Struct. Res. 1986, 95, 189-195
43． FuKamizo T, Kramer K J. Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid. Insect Biochem., 1985, 15, 141-145．
44． Fukamizo T, Kramer K J.Effect of 20-hydroxyecdysone on chitinase and β-N-acetyglucosaminidase during the larval-pupal transformation in Manduca sexta. Insect Biochem, 1987,17,547-550．
45． Gettig R R, McCarthy W J, Genotypic among wild isolates of Heliothis spp nuclear polyhedrosis viruses from different geographical regions. Viology, 1982, 117,245-252．
46． Hawtin Rachael E, Arnold Kevin, Ayres Martin D, et al., Identification and Preliminary Characterization of a Chitinase Gene in the Autographa californica Nuclear Polyhedrosis Virus Genome. Virology, 1995, 212,673-685．
47． Hawtin, R E , Zarkowska T, et al., liquefaction of Autographa californica nucleopolyhedrovirus infected insects is dependent on the intergrity of virus encoded chitinase and cathepsin genes. Virology ,1997,238,243-253．
48． Huang Xin, Zhang Hong, Zen Kuo-Chen et al., Ilomology modeling of the insect chitinase catalytic domain oligosaccharide complex and the role of a putative active site tryptophan in catalysis. Insect biochemistry and molecular biology, 2000,30,107-117．
49． Jeffrey M, Slack, John Kuzio and Peter Faulkner, 1995, Characteri-zation of cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus, Journal of General Virology, 76,1091-1098．
50． Jones J D G, Grady K L,Suslow T V. Isolation and charactera-tion of genes encoding two chitinase enzymes from Serratia marcescens.EMBO J, 1986, 5,467-473．
51． Kim M G, Shin S W, Bae K S et al., Molecular cloning of chitinase cDNAs from the silkworm, Bombyx mori and the fall webworm, Hyphantria cunea. 1998,28, 163-171．
52． Knell J D , Summers M D . A physical map of the Heliothis zea SNPV genome. Journal of general virology, 1984, 65, 445-450．
53． Kramer K J, Corpuz L, et al., Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta. Insect Biochemistry and Molecular Biology,1993, 23,691-701．
54． Kramer K J，Muthukrishnan．lnsect chitinase molecular biology and potential use as biopesticides．Insec t biochemistry and molecular biology．1997，27，11， 887-900
55． Le Hoa T，Wu Tiepiao，Robertson A，Bulach D，et al．，GeneticallY variable triplet repeats in a RINGfinger ORF of Helicoverpa species baculo-viruses，Virus Research，1997，49，67-77．
56． Luckow V A，Lee S C，Barry G F，et al．，Efficient generatjon of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagatod in Escherichia coli． Journal general virology，1993，4566-4579．
57． Martens J W M，Honee G，Zui dema D．Insecticidal acti vity of a bacterial crystal protein expressed by a recombinant baculovirus in insect cells．Appt Environ Microbiol，1990，P．2764-2770．
58． Martin D A，Stephen C H，John Kuzio，Miguel Lopez-Ferber，and Robert D Possee， The complete DNA sequence of Autographa californica Nuclear Polyhedrosis Virus．Virology，1994，202，586-650．
59． Michael D Tomalski，,Jianguo Wu and Lois K Miller，The Location，Sequence， Transcription and ReguIati on of a Baculovirus DNA Polymerase Gene，Virolgy， 1988，167，591-600．
60． Miyashita K，Fujii T．Nucleotide sequence and analysis of a gene(chiA)for a chitinase from Streptomyces lividans ．Biosci Biotechnol Biochem 1993，57，1691-1698．
61． Monsma S A，Oomens，Blissard G W，The gp64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection， Journal of General Virology，1996，70，4607-4616．
62． Perrakis A，Tews I，Dauter Z，et al．，Crystal structure of bacterial chitinase at 2． 3A resolution．Structure，1994，2，1169-1180．
63． Sasaki Y，Uchiumi Y，Hirai N，et al．，Purification and characterization of Bombyx mori chitinase．InsectL bjochemistry and molecular biology，1997，27． 9，757-767．
64． Shapira R，et al．，Control of plant diseases by chitinase expression from cloned DNA in E. coil．PhytopaLhology，1989，79，1246-1249．
65． Shen Z C, Lorena M J. Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. 1997,272,46,28895-28900．
66． Shi J By, Thomas C J, et al., The expression of a baculovirus-derivecl chitinase gene increased resistance of tobacco cultivars to brown spot(Alternaria alternata) 2000, Ann. appl. Biol , 136,1-8
67． Shih-win Ma, Bartholomew G Corsaro , et al. , Cloning and Sequence Analyses of a p40 Structural Protein Gene of Helicoverpa zca Nuclear Polyhedrosis Virus[J]. Viroi , 1993, 192, 224-223．
68． Slack J M , Kuzio J, Faulkner P. Characterization of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus. Journal of general virology, 1995,76,1091-1098．
69． St-Leger R J,Cooper R M,Charnley A K.Cuticle Degrading enzymes of entomopathogenic fungi:regulation of production of chitinolytic enzymes. J. Gen. Microbiol., 1986, 132, 1509-1517．
70． Sumiko Gomi, Kei Majima , Susumu Maeda, Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus, Journal of General Virology ,1999, 80, 1323-1337．
71． Susumu Maeda, Takashi Kawai et al., Production of human-interferon in sildworm using a baculovirus vector, Nature, 1985, 315, 13, 592-594．
72． Svitil A L, Kirchman D L. A chitin-binding domain in a marine bacterial chitinase and other microbial chitinase, implications for the ecology and evolution of 1, 4-β-glycanases. Microbiology. 1998, 144, 1299-1308．
73． Takahisa Ikegami, Terumasa Okada, et al., Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase Al,The Journal of biological chemistry, 2000, 279,18,13654-13661．
74． Thomas C J.Helen L Brown, et al., Localization of a Baculovirus Induced Chitinase in the Insect Cell endoplasmic reticulum . Journal of virology, 1998,72, 12,10207-10212．
75． Thomas C J, Gooday G W, King L A , et al., Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chiA gene. Journal of general virology, 2000 , 81,1403-1411．
76． Watanabe T，Suzuki K，Oyanagl W． Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed i ts evolutionary realationship to Serratia chitinase and to a type Ⅲ units of fibronectin．J Biol Chem，1990，265，15659-15665．
77． Watanabe T，Oyanagi W，Suzuki K，et al．，Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enyme to other prokaryotic chitinase and cl aSS Ⅲ plant chitinases．Journal of bacteriology， 1992，174． 2． 408-414．
78． Watanabe T，Kobori K，Miyashi ta K，et al．，Identification of Glutamic acid 204 in chitinase A1 of Baci11us circulans WL-12 as essential rel idues for chitinase activity．The journal of biological chemistry，1993，268，18567-18572．
79． Whitford M，Faulkner P．Nucleotide sequence and transcriptional analysis of a gene encoding gp41，a structural glycoprorein of the baculovirus Autographa californica nuclear polygedrosis virus[J]．J．Virol，1992a，66，4763-4768．
80． Williams G V，Rohel K Z，Kuzio J，et al．，A cytopathological investi-gation of Autographa californica nuclear polyhedrosis virus p10 gene function using insertion／deletion mutants．Journal of General virology，1989，70，187-201．
81． Wonkyung Kang，Michael Tristem，et al．，Identification and characterization of the Cydia pomonella granulovirus catkepsin and chitinase genes,Journal of General Virology，1998，79，2283-2292．
2．陈三凤，刘德虎，李季伦．植物几丁质酶的结构、基因及其表达．生物工程进展，1998，18(2)：33～36
3．华中师范学院生物系昆虫病毒研究室，棉铃虫核型多角体病毒的组织病理研究，昆虫学报，1978，21(2)：131-136
4．华中师范学院生物系昆虫病毒研究室组织培养组．棉铃血核型多角体病毒在离体细胞内复制，华中师范学院学报(自然科学版)，1979，(2)：18-21
5．华中师范学院生物系昆虫病毒组、荆州地区微生物站，棉铃虫核型多角体病毒的应用辅助剂的试验，华中师范学院学报，1977(1)：75-80
6．刘晨光，陈西广，刘万顺，几丁质酶Hevamine的三级结构和催化机理．生命的化学，1999，19(2)：72～74
7．卢圣栋主编 现代分子生物学实验技术 高等教育出版社
8．孟海燕，陈正华．几丁质酶及其研究进展．生命科学研究，1998，2(3)：163～176
9．欧阳石文，冯兰香，赵丌军．几丁质酶的三级结构和催化机制．生命的化学，2001，21(2)：131～133
10．彭辉银等，中国棉铃虫核型多角体病毒几丁质酶基因的定位与克隆，中国病毒学，1998，13(2)：140-142
11．彭秀玲，袁汉英等编著，基因工程实验技术(第二版)．湖南科学技术出版社
12．蒲蛰龙主编，昆虫病理学，1992，广东科技出版社
13．裘卫等，棉铃虫核型多角体病毒在同源和异源寄主细胞系内复制及其感染性比较，病毒学报，1991，7(2)：153-157
14．孙建新，两种亚型VEGF-B DNA的克隆及在昆虫细胞中的表达研究，1998，博士学位论文．上海生物化学研究所
15．孙修炼等，棉铃虫核型多角体病毒四个分离株的比较研究．中国病毒学，1994，9(4)：309-319
16．汪家政，范明主编，蛋白质技术手册，2000，科学出版社
17．王根，张传溪，金伟，等，棉铃虫核多角体病毒多角体蛋白基因的克隆和核苷酸序列分析[J]，病毒学报，1997，13(1)：82-86
18．王根，张传溪，金伟，等，棉铃虫核多角体病毒限制酶酶切图谱和多角体蛋白基因定位[J]．环境与应用生物学报，1996，2(4)：387-392
19．王根，张传溪，金伟，胡萃，吴祥甫，棉铃虫核型多角体病毒基因组限制性内酶切分析及其多角体基因的定位．应用与环境生物学报
20．王根，张传溪等，中国棉铃虫核型多角体病毒DNA聚合酶基因的克隆和序列分析，中国病毒学，1998，13(1)：77-82
21．吴青君、张文吉等，昆虫几丁质酶及其在植物保护中的应用．2000，37(5)：314-317
22．夏国庆，董志扬等，几丁质酶产生菌的筛选和酶的一般性质．农业生物技术学报，1997，5(1)：79～82
23．张传溪 昆虫杆状病毒Ph、PK基因分析和人EPO基因在杆状病毒-昆虫系统中的表达，1997，博士学位论文，浙江大学
24．张传溪等，HaSNPV 蛋白激酶基因的核苷酸序列研究，生物化学与生物物理学报，1997，29(3)：322-326
25．张传溪等，棉铃虫核型多角体病毒 HindⅢ-K 片段的核苷酸序列及其分析，中国病毒学，1998，13，(2)：142-149
26．张传溪等，EGFP 基因在粉蚊夜蛾中的高效表达，动物学研究，1999，20(6)：468-470
27．张传溪，贡成良等，杆状病毒包涵体蛋白基因的进化，浙江农业大学学报，1999，25(1)：69—76
28．张传溪，武家才．棉铃虫核型多角体病毒基因组结构及p10基因．生物化学与生物物理学报，生物化学与生物物理学报，2001，33(2)：179-184
29．钟江等，棉铃虫细胞系SFE-IIA—8212的克隆化及对HaSNPV 受纳性提高的克隆系的建立，复旦学报(自然科学版)，1994，33(2)：148-156
30．朱雪峰，陈崇顺，郁志芳．植物几丁质酶的生物功能．生命的化学，2000，20(1)：36～37
31．Bonneng B c &Hammock B D,基因工程杆状病毒杀虫剂的发展与潜力．国外农学-蚕业，1993(4)：12-21
32．J 萨姆布鲁 E F，弗里奇 T 曼尼阿蒂斯著，金冬雁，黎盂枫等译，分子克隆实验指南
[M].1993，科学出版社
33． Bhushan-Bharat; Hoondal-G-S Effect of fungicides, insecticides and allosamidin on a thermostable chitinase from Bacillus sp. BG-11． June, World-Journal-of-Microbiology-and-Biotechnology. 1999,15 (3) , 403-404．
34． Blaak H ,Schnellmann J ,Walter S , Henrissat B. Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, pupative protein domains and relationship to other chitinase. Eur J Biochem . 1993,214,659-669．
35． Chaerchomsri S,Ikeda M, Kobayashi M. Nucleotide sequence of the Lymantria dispar nuclear ployhedrosis virus DNA polymerase gene region, Journal of General Virology, 1992, 73,3177-3183．
36． Chen X W, Sun X L, HU Z M et al., Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide. Journal of invertebrate pathology, 2000, 76,140-146．
37． Chernin L. S., Fuente de la, Sobolev L. V. Molecular cloning, structural analysis, and expression in Escherichia coli. of a chitinase gene from Enterobacter agglomerans. 1997,63,834-839．
38． Cowan P, Bulach D, et al. , Nucleotide Sequence of the Polyhedrin Gene Region of Helicoverpa zea single nucleocapsid nuclear polyhedrosis virus: Placement of the Virus in Lepidopteran nuclear polygidrosis virus groupⅡ, Journal of General virology, 1994,75,3211-3218．
39． Ding X.Manduca chitinase-mediated resistance to tobacco budworm(Heliothis mirescens)and tobacco hornworm (Manduca scxta) larvae in transgenic tobacco plants. Ph. D. Dissertation, Kansas State University, Manhattan. 82 p.
40． Ding X, Gopalakrishnan B, Johnson L B, White F F, Wang X, Morgan T D, Kramer K J, et al.,Insect resistance of transgenic tabacco expressing an insert chitinase gene. Transgen. Res. 1997,75,3211-3218．
41． El-Sayed G N,Coudron T A, Ignoffo C M.Chitinolytic activity and vurulence associated with native and mutant isolates of the entomopathogenic fungus, Nomuraea ileyi.J. Invertr. Pathol., 1989,54,394-403．
42． Fraser M J, Ultrastructural observations of virion maturation in Autographa californica nuclear polyhedrosis virus infected Spodop-tera frugiperda cell cultures [J]. J. Ultrast. Mol. Struct. Res. 1986, 95, 189-195
43． FuKamizo T, Kramer K J. Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid. Insect Biochem., 1985, 15, 141-145．
44． Fukamizo T, Kramer K J.Effect of 20-hydroxyecdysone on chitinase and β-N-acetyglucosaminidase during the larval-pupal transformation in Manduca sexta. Insect Biochem, 1987,17,547-550．
45． Gettig R R, McCarthy W J, Genotypic among wild isolates of Heliothis spp nuclear polyhedrosis viruses from different geographical regions. Viology, 1982, 117,245-252．
46． Hawtin Rachael E, Arnold Kevin, Ayres Martin D, et al., Identification and Preliminary Characterization of a Chitinase Gene in the Autographa californica Nuclear Polyhedrosis Virus Genome. Virology, 1995, 212,673-685．
47． Hawtin, R E , Zarkowska T, et al., liquefaction of Autographa californica nucleopolyhedrovirus infected insects is dependent on the intergrity of virus encoded chitinase and cathepsin genes. Virology ,1997,238,243-253．
48． Huang Xin, Zhang Hong, Zen Kuo-Chen et al., Ilomology modeling of the insect chitinase catalytic domain oligosaccharide complex and the role of a putative active site tryptophan in catalysis. Insect biochemistry and molecular biology, 2000,30,107-117．
49． Jeffrey M, Slack, John Kuzio and Peter Faulkner, 1995, Characteri-zation of cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus, Journal of General Virology, 76,1091-1098．
50． Jones J D G, Grady K L,Suslow T V. Isolation and charactera-tion of genes encoding two chitinase enzymes from Serratia marcescens.EMBO J, 1986, 5,467-473．
51． Kim M G, Shin S W, Bae K S et al., Molecular cloning of chitinase cDNAs from the silkworm, Bombyx mori and the fall webworm, Hyphantria cunea. 1998,28, 163-171．
52． Knell J D , Summers M D . A physical map of the Heliothis zea SNPV genome. Journal of general virology, 1984, 65, 445-450．
53． Kramer K J, Corpuz L, et al., Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta. Insect Biochemistry and Molecular Biology,1993, 23,691-701．
54． Kramer K J，Muthukrishnan．lnsect chitinase molecular biology and potential use as biopesticides．Insec t biochemistry and molecular biology．1997，27，11， 887-900
55． Le Hoa T，Wu Tiepiao，Robertson A，Bulach D，et al．，GeneticallY variable triplet repeats in a RINGfinger ORF of Helicoverpa species baculo-viruses，Virus Research，1997，49，67-77．
56． Luckow V A，Lee S C，Barry G F，et al．，Efficient generatjon of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagatod in Escherichia coli． Journal general virology，1993，4566-4579．
57． Martens J W M，Honee G，Zui dema D．Insecticidal acti vity of a bacterial crystal protein expressed by a recombinant baculovirus in insect cells．Appt Environ Microbiol，1990，P．2764-2770．
58． Martin D A，Stephen C H，John Kuzio，Miguel Lopez-Ferber，and Robert D Possee， The complete DNA sequence of Autographa californica Nuclear Polyhedrosis Virus．Virology，1994，202，586-650．
59． Michael D Tomalski，,Jianguo Wu and Lois K Miller，The Location，Sequence， Transcription and ReguIati on of a Baculovirus DNA Polymerase Gene，Virolgy， 1988，167，591-600．
60． Miyashita K，Fujii T．Nucleotide sequence and analysis of a gene(chiA)for a chitinase from Streptomyces lividans ．Biosci Biotechnol Biochem 1993，57，1691-1698．
61． Monsma S A，Oomens，Blissard G W，The gp64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection， Journal of General Virology，1996，70，4607-4616．
62． Perrakis A，Tews I，Dauter Z，et al．，Crystal structure of bacterial chitinase at 2． 3A resolution．Structure，1994，2，1169-1180．
63． Sasaki Y，Uchiumi Y，Hirai N，et al．，Purification and characterization of Bombyx mori chitinase．InsectL bjochemistry and molecular biology，1997，27． 9，757-767．
64． Shapira R，et al．，Control of plant diseases by chitinase expression from cloned DNA in E. coil．PhytopaLhology，1989，79，1246-1249．
65． Shen Z C, Lorena M J. Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. 1997,272,46,28895-28900．
66． Shi J By, Thomas C J, et al., The expression of a baculovirus-derivecl chitinase gene increased resistance of tobacco cultivars to brown spot(Alternaria alternata) 2000, Ann. appl. Biol , 136,1-8
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