Isolation of p10 gene fromBombyx mori nuclear polyhedrosis virus and study of its promoter activity in recombinant baculovirus vector system

A homologue ofAutographa californica NPV (AcNPV) p10 gene was identified and cloned fromBombyx mori NPV (BmNPV). BmNPV p10 gene encodes truncated protein of 70 amino acid residues that lacks carboxyl terminus comparing with the p10 protein encoded by AcNPV. The putative TATA box sequence and the ATAAG motif which is the consensus sequence of baculovirus very late promoter were conserved. A transfer vector, pBNT1, which includes the p10 promoter region of BmNPV for foreign gene expression was constructed. By using pBNT1, a recombinant BmNPV, Bmp10-Luc, in which the p10 gene was replaced by the firefly luciferase gene, was obtained. We also obtained another recombinant virus, BmPH-Luc, in which the polyhedrin gene was replaced by the luciferase gene. The luciferase activity detected in BoMo-15AIIc insect cells infected with Bmp10-Luc was approximately 50% of that infected with BmPH-Luc, suggesting that although both the p10 and polyhedrin promoters of BrnNPV are effective in high-level expression of foreign gene, the p10 promoter is not so strong as the polyhedrin promoter.     

以核多角体病毒为载体在家蚕中生产外源蛋白

以家蚕核多角体病毒(BmNPV)为载体,在家蚕幼虫或家蚕培养细胞系中表达的外源基因越来越多,其表达的产物已涉及到医用药物、医疗诊断、疫苗生产、生物防治等诸多领域,文章就BmNPV的特性及其基因组构造,多角体蛋白基因的特性,重组BmNPV的构建及其在家蚕幼虫体内和细胞系中的表达,BmNPV-家蚕表达系统的外源蛋白生产效率及其应用等各个方面作了全面、系统的综述.     

High-level expression and improved folding of proteins by using the vp39 late promoter enhanced with homologous DNA regions

Some recombinant proteins expressed by baculovirus expression vector systems (BEVS) aggregate because the BEVS can produce large amounts of protein late during infection, when post-translational modification and protein quality control mechanisms are inactive. For expression during earlier stages than that driven by the polyhedrin (polh) very late promoter, transfer vectors were generated in which this promoter was replaced with a green fluorescent protein (GFP) gene controlled by a vp39 late promoter modified to contain HR3, one of the homologous DNA regions (HRs) of Bombyx mori nuclear polyhedrosis virus (BmNPV). The rise times of the fluorescence of GFP expressed by using recombinant viruses carrying the modified vp39 promoter were earlier than those associated with either the polh promoter or the native vp39 promoter lacking HR3. In transient expression assays, the vp39 late promoter in transfer vectors behaved like a delayed-early promoter, and was enhanced by HR3, and required IE-1 protein and various viral gene products encoded on both sides of BmNPV polh. When the vp39 promoter with HR3 was used, the aggregation of several foreign proteins expressed by the BEVS was markedly decreased. This study provides a new option for the expression of sufficiently quality-controlled proteins by using the vp39 promoter and HR3 in BEVS early in baculovirus infection, when the infection has caused little damage in the host cells.     

杆状病毒凋亡抑制基因IAP1促进家蚕CyclinB的核内积累

[目的]家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是生产上危害最严重的病原之一。BmNPV感染BmN-SWU1细胞将细胞周期阻滞于G2/M期。CyclinB是调控细胞周期G2期向M期转换的重要细胞周期蛋白。因此,研究BmNPV感染后CyclinB变化对解析病毒调控细胞周期的机制具有重要意义,同时探究这个过程中与CyclinB互作的病毒蛋白,可为构建家蚕转基因品系提供分子靶标。[方法]qRT-PCR检测BmNPV感染后BmCyclinB的表达变化;免疫荧光观察病毒感染前后BmCyclinB的定位变化,通过细胞质细胞核蛋白分离实验验证。免疫共沉淀钓取与BmCyclinB互作的病毒蛋白。BmNPV感染期间敲除BmNPV IAP1观察BmCyclinB的入核比例。[结果]BmNPV感染后BmCyclinB转录水平下调。BmNPV感染前BmCyclinB主要定位于细胞质,而感染后主要定位于细胞核。BmNPV感染BmN-SWU1细胞后促进BmCyclinB在核内积累。共钓取了7个与BmCyclinB互作的病毒蛋白,免疫共沉淀和细胞共定位证明BmNPV IAP1与BmCyclinB之间存在相互作用。敲除BmNPV IAP1后BmCyclinB进入细胞核的数量显著减少。[结论]BmNPV IAP1可通过与BmCyclinB互作,促进BmCyclinB在核内积累。     

Construction of the Bac-to-Bac system of <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae. Foundation items: 973 (2003CB114202); Programme Strategic Scientific Alliances between China and the Netherlands (2004CB720404); National Natural Fundation of China project (30630002)     

Efficient Protein Expression in <Emphasis Type="Italic">Bombyx mori</Emphasis> Larvae of the Strain d17 Highly Sensitive to <Emphasis Type="Italic">B. mori</Emphasis> Nucleopolyhedrovirus

The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a “factory” for large-scale expression using the BmNPV bacmid system.     

家蚕ADP/ATP转运酶(BmANT)抑制BmNPV增殖作用机制研究

【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)隶属于杆状病毒,需要借助宿主细胞能量代谢进行自身增殖复制。家蚕ADP/ATP转运酶(Bombyx mori ADP/ATP translocase,BmANT)是线粒体转运蛋白,在BmNPV感染条件下和家蚕热休克蛋白60(heatshockprotein60,BmHSP60)具有直接的相互作用。因此,鉴定Bmant基因在BmNPV感染过程中的功能特征,有助于解析杆状病毒劫持宿主细胞因子促进自身增殖复制机制,完善杆状病毒和宿主相互作用网络。【方法】通过结构域预测BmANT蛋白的结构特征,荧光定量PCR分析Bmant基因在BmNPV感染后的变化特征;并过表达BmANT检测其对病毒DNA复制和病毒蛋白表达变化影响;进一步在转录水平分析Bmant和Bmhsp60基因的调控关系;最后通过流式细胞术等技术鉴定Bmant和Bmhsp60基因共同调控BmNPV增殖复制的机制。【结果】SMART软件预测显示BmANT包含3个线粒体载体结构域,BmNPV感染24 h后Bmant基因持续下调表达。过表达Bmant基因能够显著抑制BmNPV DNA的复制和VP39蛋白表达。荧光定量PCR分析显示Bmant和Bmhsp60基因具有相互拮抗作用,能够相互抑制转录。Bmant和Bmhsp60共同过表达分析显示,BmANT和BmHSP60共同作用BmNPV能够抑制病毒的增殖复制。【结论】结果表明,BmANT是一个线粒体载体蛋白,具有显著的抗病毒作用,能够下调Bmhsp60基因表达,并抑制BmNPV增殖复制。     

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > − 3 > − 8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60–72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in ‘glowing silkworms’. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.     

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50–100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.     

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.     

家蚕核型多角体病毒lef-11基因RNA干扰策略的优化

【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)lef-11基因作为杆状病毒高度保守的晚期表达因子之一,对病毒晚期基因的表达极其重要。因此对lef-11基因进行有效RNA干扰将提高宿主细胞对BmNPV的抗性。【方法】本文基于经典的sh-RNA-loop骨架及家蚕内源性的miRNA骨架,构建以BmNPV lef-11基因为靶标的干扰载体pIZ-shRNA1、pIZ-shRNA2和pIZ-Ds RedamiR279;分别构建基于A4、IE1、IE1-295、IE2、IE2-339、hr3A4和hsp70启动子驱动的干扰载体,用以评价不同启动子驱动的干扰载体的抗病毒效果,并对干扰载体进行优化。【结果】首先,本文通过比较miRNA-based和shRNA-based RNAi载体对同一靶基因同一位点的干扰效率,发现pIZ-Ds Red-amiR279对BmNPV lef11基因的干扰效率超过90%,远优于shRNA-based RNAi载体的干扰效果;其次,通过对干扰载体进行优化,发现IE1启动子的效果最优,由其驱动的pIZ-Ds Red-milef11干扰载体也是本研究中的最优干扰载体,对病毒的增殖抑制效果最为明显。【结论】对目的基因的干扰效果并非启动子的活性越高、miRNA积累得越多就越好,只有综合考虑干扰片段的选择、启动子的活性以及靶基因自身的功能等多方面的因素,才能提高干扰效率,达到干扰目的。     

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.     

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insectbaculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect,A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm,Bombyx mori, which is wellknown for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for largescale industrial expression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in commonuse cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the production efficiency of the bFGF recombinant protein.     

斜纹夜蛾核多角体病毒gp37及其邻近序列的克隆及分析

从斜纹夜蛾核多角体病毒(Spodoptera litura nucleopolyhedrovirus, SpltMNPV)基因组中克隆了gp37基因.分子生物学软件分析表明,在gp37基因内部存在糖基化位点.含有晚期表达基因的启动子序列(ATAAG),推测为晚期表达的病毒膜糖蛋白基因.通过GP37蛋白的同源性比较,绘制了以gp37基因为基础的杆状病毒进化树, 发现与以多角体蛋白基因（polyhedrin）为基础所绘制的杆状病毒分子进化树有较大差异.以polyhedrin基因为基础绘制的杆状病毒分子进化树将家蚕核多角体病毒（Bombyx mori nucleopolyhedrovirus, BmNPV）与杆状病毒代表种——苜蓿丫纹夜蛾核多角体病毒（Autographa californica multinucleocapsid nucleopolyhedrovirus, AcMNPV）分开,根据gp37基因分析则将二者归到同一分枝,这与以egt基因为基础绘制的杆状病毒分子进化树结果一致.     

The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors

A system is described for the rapid generation of Bombyx mori nucleopolyhedrovirus (BmNPV)-based expression vectors. A series of novel BmNPV genomes, that include a mini-F replicon and therefore can be maintained in Escherichia coli, have been generated. These genomes lack a portion of the essential ORF1629 gene and cannot replicate independently in insect cells. However, they can be used as parental genomes for the generation of expression vectors by cotransfection with a transfer plasmid that includes an intact ORF1629. Only recombinant viruses that have acquired the ORF1629 gene from the transfer vector, and have therefore also acquired the foreign gene of interest, can replicate after cotransfection. Parental genomes with and without a polyhedrin gene are described, enabling the generation of occlusion-positive and occlusion-negative recombinant viruses. Occlusion-positive expression vectors enable the oral infection of B. mori larvae and can therefore be used for the mass production of a foreign protein in infected insects.