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

【作者】 武家才；

【导师】 张传溪；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2001， 硕士

【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。HaSNPVp40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更多还原

【Abstract】 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.更多还原