Cloning And Expression Of Helicoverpa Armigera Single Nucleopolyhedrovirus Superoxide Dismutase Gene

Baculovirus is one of the most important pathogenic microorgisms which infect Lepidopteran insects. They are attractive biological agents for the control of agriculturally important insect pests.The research to HaSNPV genes is useful for understanding the functions of these genes in the baculovirus living cycle. Superoxide Dismutase(SOD) is a kind of important antioxidant enzyme that deletes (V in cell. The coding region of superoxide dismutase was amplified by using PCR method from Helicoverpa armigera single enveloped nucleocapsid nucleopolyhedrovirus (HaSNPV) Cl genome. The PCR product was cloned into pGEM-T easy vector and then was sequenced. The HaSNPV SOD nucleic acid sequence is analyzed, The alignment of HaSNPV SOD amino acid sequence with those of other 15 reported baculovirus SODs is completed with the Genetyx . At the same time, the evolutionary relationships to those of other baculoviruses were analyzed with UPGMA and NJ program. HaSNPV sod gene has been expressed in E.coli and insect cell, respectively. Main results of this dissertation included three aspects. 1.Sequence analysis of HaSNPV sod geneThe analysis indicated that HaSNPV sod gene was localized 100,487-100,966 bp downstream of the polyhedrin gene and transcribed in the same direction.The coding region was amplified by using PCR method from Helicoverpa armigera single enveloped nucleocapsid nucleopolyhedrovirus (HaSNPV) Cl genome and sequenced. The results showed that the cloned gene sequence in the study was identical to the reported HaSNPV Cl sod sequence. An open reading frame of 480 nucleotides encoding a putative 159 amino acid polypeptide with predicted molecular mass of 16.8 kDa representing the superoxide dismutase gene was determined. The content of G+C is 48.13%. A conserved motif ATAAG was identified -18 nucleotides upstream of the sod gene start codon, ATG. Amino acid sequences of 16 SOD poteins were compared by using Genetyx program. The result showed that HaSNPV sod gene was most closely related to that of HzSNPV, with 98.7% amino acid identity. HaSNPV also shared a high degree of homology with other lepidopteran NPVs, with identities above 69.1%, and with other lepidopteran GVs, with identities ranging from 53.0%-62.4%. HaSNPV SOD shared a high degree of homology with human SOD, with identities 49%. HaSNPV SOD has identical conserved amino acid polypeptide as human SOD.It was deduced that HaSNPV SOD is a kind of Cu/Zn-SOD from the amino acid sequences identities and the conserved motifs. The evolutionary relationships to those of other baculoviruses were analyzed with UPGMA and NJ program.The results showed that NPV SOD and GV SOD belong to different branches. Human SOD belongs to other branch. HaSNPV SOD is most closely related to HzSNPV SOD.2.Expression of HaSNPV sod gene in E.coliThe coding region of HaSNPV sod gene was cloned into expression vector pETBlue-2. The recombinant expression plaimid pETBlue-2/HaSNPV SOD was constructed and transformed into DE3(BL21). The gene was then expressed in the transformed E.coli with the induction of IPTG. The result of SDS-PAGE analysis showed that the expression content of SOD is 37% of the total recombinant protein. The enzyme activity of HaSNPV SOD was assayed by Pyrogallol autooxidation method. The result showed that the revised enzyme activity of the expressed product in the total soluble protein was 694 U/mg.3.Expression of HaSNPV sod gene in insect cellThe coding region of HaSNPV sod gene was inserted into the transfer plasmid pBacHTeGFP to generate the recombinant transfer plasmid pBacHTeGFP/SOD. The transfer vector is under the control of strong ph gene promoter. His tag and eGFP gene were inserted into the plasmid between the promoter and MCS.Then the recombinant transfer plasmid pBacHTeGFPT/SOD was transformed into DH10 with Bacmid . The recombinant virus genome Bacmid- sod was extracted. The PCR reaction confirmed that sod gene had been correctly inserted into the target position in the virus genome. Transfection of Tn-5B1-4 cells with recombinant Bacmid D...