Construction of the Bac-to-Bac System of Bombyx mori 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.     

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)     

转座子介导的多角体基因表达及提高病毒粒子的感染效率

现行的杆状病毒表达外源基因的方法是将外源基因取代病毒中的多角体基因,因而得到的重组杆状病毒感染活体时不能经口感染,只能进行针刺注射,效率低且易引起活体感染其他疾病。将家蚕核型多角体病毒(Bombyx mor inucleopolyhedrovirus,BmNPV)中的多角体基因(polyhedrin,poly)及其启动子片段克隆到转座子载体pigA3GFP中,将其与辅助质粒pHA3PIG利用脂质体介导法导入家蚕细胞中,经过多次筛选获得稳定的转基因家蚕细胞。之后先将BmPAK6(含LacZ)及BmGFP(含GFP)重组病毒分别感染转基因细胞,再将得到的重组病毒经口感染5龄家蚕幼虫。结果显示,重组杆状病毒可以经口感染家蚕幼虫。这些研究表明来自于转基因家蚕细胞的poly基因表达产物可以提高重组杆状病毒经口感染家蚕率,为解决杆状病毒表达系统中重组病毒不能经口感染家蚕幼虫的问题提供新思路。     

A novel way to purify recombinant baculoviruses by using bacmid

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.     

High-frequency homologous recombination between baculoviruses involves DNA replication

We determined the frequency of DNA recombination between Bombyx mori nucleopolyhedroviruses (BmNPVs) and between BmNPV and the closely related Autographa californica NPV (AcMNPV) in BmN cells, Sf-21 cells, and larvae of Heliothis virescens. The BmN cells were coinfected with two BmNPVs, one with a mutation at the polyhedrin gene (polh) locus and a second carrying a lacZ gene marker cassette. Eleven different BmNPV mutants carrying the lacZ gene marker at various distances (1.4 to 61.7 kb) from polh were used for the coinfections. The Sf-21 cells and larvae of H. virescens were coinfected with wild-type AcMNPV and 1 of the 11 lacZ-marked BmNPV mutants. In BmN cells, high-frequency recombination was detected as early as 15 h postcoinfection but not at 12 h postcoinfection. At 18 h postcoinfection, the mean frequency of recombination ranged between 20.0 and 35.4% when the polh and lacZ marker genes were separated by at least 9.7 kb. When these marker genes were separated by only 1.4 kb, the mean frequency of recombination was 2.7%. In BmN cells, the mean recombination frequency between two BmNPVs increased only marginally when the multiplicity of infection of each virus was increased 10-fold. In Sf-21 cells and the larvae of H. virescens, the recombination frequency between BmNPV and AcMNPV was 相似文献   

拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体,与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN,经200μg/ml zeocin抗生素筛选一个月,成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒,其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染,结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主,却并不在宿主体内形成包涵体,从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题,通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。     

异源多角体蛋白对家蚕核型多角体病毒粒子的包装

利用PCR方法从AcMNPV基因组DNA中分离出多角体蛋白基因 ,将该扩增片段克隆到转移载体pBacPAK8中 ,得到重组转移载体pOAc。将该质粒DNA与线性化的Bm BacPAK6病毒基因组DNA共传染BmN细胞 ,得到了能形成多角体且不产生蓝色空斑的重组病毒hp BmNPV。纯化该重组病毒的多角体颗粒 ,并对多角体蛋白、病毒核酸及多角体病毒颗粒进行分析 ,发现AcMNPV的多角体蛋白能在家蚕细胞中大量表达且能在细胞内识别家蚕核型多角体病毒并组装成多角体颗粒 ;病毒基因组DNA因部分交换 ,其酶切行为发生了相应的变化 ;电镜观察发现经AcMNPV多角体蛋白包装的家蚕核型多角体病毒的多角体颗粒大小为1 2 μm～ 2 9μm ,明显小于野生型家蚕核型多角体病毒的多角体颗粒     

失活 ChiA 和 v-cath 基因的重组BmNPV 表达 hGM-CSF

利用 DNA 同源重组技术使家蚕核型多角体病毒的多角体蛋白基因取代 v-cath 、 ChiA 两基因的部分编码区域和共同的启动子区域,获得了 v-cath 、 ChiA 两基因同时失活的、可形成多角体的、并能表达人粒细胞 - 巨噬细胞集落刺激因子的重组病毒 BmNPVpolh+CP－ChiA－GMCSF+. 研究结果显示： v-cath 、 ChiA 两基因的失活不影响病毒的复制及多角体的形成,但感染细胞的存活时间比野生病毒感染的多 2 天,可明显改善外源基因在培养细胞和家蚕中的表达水平;感染 BmNPV polh+CP－ChiA－GMCSF+的家蚕幼虫体表保持正常,无野生病毒感染后引起的破皮流脓现象 .     

Comparative studies of Bombyx mori nucleopolyhedrovirus chitinase and its host ortholog, BmChi-h

Baculovirus-encoded chitinases (V-CHIAs) were first proposed to be acquired from a bacterium via horizontal gene transfer. However, we have recently reported that lepidopteran hosts also encode v-chiA orthologs. Here we describe comparative studies of Bombyx mori nucleopolyhedrovirus (BmNPV) chitinase and its host ortholog, BmChi-h. We constructed recombinant BmNPVs in which native and modified forms of BmChi-h were driven under the polyhedrin promoter and the authentic v-chiA was deleted. Western blot analysis indicated that BmCHI-h was rapidly secreted from virus-infected BmN cells whereas BmNPV CHIA was localized within the virus-infected cells; probably because of the presence of a C-terminal endoplasmic reticulum retention motif on BmNPV CHIA. Enzymological studies showed that BmNPV CHIA was able to retain much higher chitinolytic activity under alkaline conditions. For B. mori larvae infected with v-chiA-deleted BmNPV, the terminal liquefaction of dead larvae and the activation of baculovirus-encoded cysteine protease were not observed, and the introduction of BmChi-h did not rescue these defects. Our findings show that BmNPV chiA possesses unique features that are not shared by host orthologs, which may reflect functional specialization of baculovirus chitinases.     

Enhanced production of secretory beta1,3-N-acetylglucosaminyltransferase 2 fusion protein into hemolymph of Bombyx mori larvae using recombinant BmNPV bacmid integrated signal sequence

The enhanced secretion of beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) fusion protein into the hemolymph of Bombyx mori larvae was studied using a recombinant B. mori nucleopolyhedrovirus (BmNPV) bacmid integrating seven signal sequences. When the BmNPV bacmid encoding the signal sequences from the silkworm B. mori bombyxin (bx) and B. mori prophenoloxidase-activating enzyme (ppae) was injected into silkworm larvae, 56.1 and 51.5mU/ml beta3GnT, respectively, were secreted into the hemolymph of silkworm larvae. For bx, 97.3% of the total beta3GnT activity was secreted into hemolymph, and only 1.1% remained in the intestines of silkworm larvae. For ppae, 90.8% of the total beta3GnT activity was secreted to the hemolymph, but 7.8% remained in the intestines of silkworm larvae. Using the BmNPV bacmid encoding bx, the amount of secreted beta3GnT was 91mug per larva, which was 2.5% of the total amount of protein in the hemolymph.     

家蚕BmNPV多角体蛋白与外源蛋白共包埋入多角体的研究

为了探索家蚕核型多角体病毒多角体的包装特性,构建了一种不形成多角体但能大量表达绿色荧光蛋白(EGFP)的重组病毒vBmBac(polh-)-5B-EGFP,将其与野生型BmNPV共同感染BmN细胞,于荧光显微镜下观察到EGFP与多角体可以在同一细胞中同时表达。从感染的BmN细胞中收集纯化多角体,观察到多角体能被激发出绿色荧光,进一步利用Western blot证实多角体中含有EGFP。上述结果表明,多角体可以将自身病毒粒子以外的其他病毒粒子的成分包装进入多角体,表明多角体的包装机制中存在非特异性识别机制。     

家蚕表达人表皮生长因子gp67信号肽融合蛋白及生物活性的研究

人表皮生长因子(hEGF), 一种由53个氨基酸残基组成的单链多肽, 具有广阔的应用前景。本文主要探讨家蚕表达人表皮生长因子gp67信号肽融合蛋白的生物活性。采用家蚕杆状病毒表达系统来表达该信号肽融合蛋白。构建了重组质粒pBacPAKS-hEGF, 将该重组质粒与线性化病毒Bm-BacPAK6 DNA共转染家蚕细胞, 筛选获得重组病毒vBacPAK-SEGF, 用vBacPAK-SEGF感染家蚕BmN细胞和五龄蚕, Western blot检测表明在家蚕细胞、五岭幼虫的血淋巴和蛹中均有约12 kD的目的蛋白表达。ELISA检测发现在家蚕细胞中的表达量为23 mg/ 106细胞, 五龄幼虫中的表达量可达到82 mg/mL血淋巴。利用小鼠成纤维细胞Balb/c3T3分析家蚕表达的hEGF信号肽融合蛋白的生物活性, 结果表明表达产物能显著促进Balb/c3T3细胞的增值。另外, 研究还发现hEGF信号肽融合蛋白可使新生ICR小鼠体重增 加, 睁眼和萌齿时间提前。本研究为进一步开发利用家蚕表达的hEGF提供理论基础。     

High level expression of a mutant (K151E, R154G) of single chain urokinase-type plasminogen activator in silkworm

A cDNA of a mutant (K151E, R154G) of single chain urokinase-type plasminogen activator (mscu-PA) was constructed to include the natural scu-PA signal peptide sequences and transferred into the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) by transfer vectors pBE284 (derived from BmNPV) and pVL1392 (from AcNPV), respectively. Both Bombyx mori (BmN) cells and silkworm larvae were infected with the two recombinant viruses. Fibrin-plate assay showed that the re-virus from pVL1392 increased the yield of mscu-PA three times compared with the re-virus from pBE284.     

Inactivation or removal of the budded particles of a nuclear polyhedrosis virus of a silkworm, Bombyx mori L. (Lep., Bombycidae)

Effective methods to inactivate or remove budded particles of a nuclear polyhedrosis virus of Bombyx mori (BmNPV) from a cell-cultured media or from host haemolymph that is infected by this virus have been developed. Two types of suspensions containing BmNPV budded virus particles, TC-100 media that cultured BmN4 cells infected by this virus and haemolymph of B. mori larvae infected by this virus, were treated by 6% (w/v) polyethylene glycol (PEG), 0.01% (w/v) chitosan, 0.05% (v/v) linoleic acid (an emulsion), and/or diethylether. Treatment by linoleic acid followed by PEG-precipitation and treatment by diethylether followed by PEG-precipitation were so effective that these treatments suppressed the viral titre of BmNPV-infected larval haemolymph from an original titre (> 10⁹ TCID₅₀ units/ml) to below a detectable limit. These methods are suggested as being potentially useful in an insect factory system; that is, a protein production system utilizing a baculovirus vector and its insect host or cultured cells on a large scale.     

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.     

Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain

Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90?% on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV.     

利用Red系统快速敲除家蚕核型多角体病毒orf60基因

用Red重组系统和最近构建的家蚕核型多角体病毒(BmNPV)bacmid在大肠杆菌BW25113中快速地敲除BmNPVorf60基因。从大肠杆菌BmDH10Bac中提取BmNPVbacmid,将其电转化到含有质粒pKD46(能表达Red重组酶)的大肠杆菌菌株BW25113中,获得了可用于BmNPV基因打靶的菌株BW25113-Bac。设计一对长63bp的引物(5′端为orf60基因的左右同源臂,长45bp;3′端长18bp,为氯霉素抗性基因(cat)的首尾序列),以pKD3质粒(含cat)为模板,PCR扩增携带orf60左右同源臂的cat,即打靶线性化片段。将该线性化片段电转入BW25113-Bac菌株,在Red重组酶的作用下,线性化片段与BmNPVbacmid中的orf60基因发生同源重组。设计3对特异引物,用PCR方法证明cat成功地替换了BmNPVorf60基因。重组bacmid DNA转染BmN细胞后,Western blot分析未检测到orf60基因的表达。     

家蚕类泛素化翻译后修饰蛋白NEDD8的cDNA克隆、表达分析和亚细胞定位

【目的】NEDD8是一种重要的蛋白质翻译后修饰蛋白,对底物蛋白的功能具有重要的调节作用。本研究旨在探索家蚕Bombyx mori中NEDD8的功能。【方法】利用RT-PCR技术,从家蚕Bm N细胞中克隆了家蚕NEDD8完整的开放阅读框。通过实时荧光定量PCR(qRT-PCR)技术检测家蚕NEDD8在不同发育阶段、5龄第3天幼虫不同组织中以及Bm NPV感染Bm N细胞后的相对表达量。通过构建GFP融合表达的重组Bm NPV(B.mori nucleopolyherovirus)感染家蚕Bm N细胞,在共聚焦显微镜下观察NEDD8在细胞中分布情况,用GFP抗体进行Western blot验证。【结果】克隆获得了NEDD8基因。序列分析表明,家蚕NEDD8高度保守,与家蚕泛素蛋白氨基酸序列一致性最高。qRT-PCR分析结果表明,NEDD8在家蚕的不同组织中均有表达,其中头部中表达量最高,其次是丝腺中,而在精巢和卵巢中表达量最低;在家蚕5龄第3天幼虫始到化蛹后第3天NEDD8的表达量开始逐渐增加,化蛾后降至低水平;在家蚕杆状病毒感染Bm N细胞的早期和极晚期NEDD8的表达量都有明显增加。GFP-NEDD8融合表达定位显示NEDD8在Bm N细胞内普遍存在,分布于整个细胞中,并且在感染48 h后存在细胞质内的聚集现象。【结论】NEDD8编码序列在物种间高度保守;NEDD8在家蚕幼虫头部中表达量最高,在化蛹阶段表达量逐渐增加;NEDD8在Bm N细胞内普遍存在并且可能与参与Bm NPV复制。本研究所得结果为进一步研究NEDD8在家蚕中的生物学功能及修饰底物蛋白的作用机制奠定了基础。     

甜菜夜蛾核多角体病毒BAC-TO-BAC外源基因表达系统的建立

用直接克隆法将miniF-lacZ-attFn7-kan 片段插入甜菜夜蛾核多角体病毒（Spodoptera exigua multicapsid nucleopolyhedrovirus, SeMNPV）〖JP〗美国分离株（SeUS1）基因组的多角体蛋白基因框内,miniF是大肠杆菌F因子复制子,携带miniF的重组病毒能够在大肠杆菌中低拷贝稳定复制,称为bacmid。由于SeUS1由不同的SeMNPV基因型组成,每个bacmid携带了一种病毒基因型,所有bacmid构成了SeUS1分离株的BAC文库。REN对111个bacmid分析表明,SeUS1分离株中除了包含具有完整SeMNPV遗传信息的基因型外,还包括不同类型的缺失基因型。将具有完整SeMNPV基因组的基因型SeBAC10转染昆虫细胞,可产生子代病毒,故SeBAC10是一种在真核细胞和原核细胞中均能复制的穿梭质粒。因为SeBAC10中多角体蛋白基因(Seph)被插入失活,将Seph作为报告基因通过位点特异性重组方式插入位于LacZ框内转座子Tn7的附着靶位点attTn7,得到重组SeBAC10 (即SeBAC10ph)转染甜菜夜蛾培养细胞Se301后,细胞出现典型的病理变化,核中出现多角体,证明SeMNPV BAC-TO-BAC外源基因表达载体系统构建成功。