Improved generation of recombinant baculovirus genomes in Escherichia coli

An improved method for the generation of recombinant baculoviruses by Tn7-mediated transposition is described. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculovirus bacmids in Escherichia coli with a mutated SacB gene. Recombinant bacmids can be generated at a frequency of ~10⁷/µg of donor vector with a negligible background. This easy-to-use and efficient pBVboost system provides the basis for a high-throughput generation of recombinant baculoviruses as well as a more convenient way to produce single viruses. The introduced selection scheme is also useful for the construction of other vectors by transposition in E.coli.     

利用杆状病毒Bac-to-Bac系统在Sf9细胞中表达真菌细胞色素P450nor

利用BactoBac杆状病毒载体表达系统将真菌细胞色素P450nor基因克隆至转移载体pFastBac1中, 得到重组质粒pFastBacP450nor, 再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中发生转座作用, 得到含P450nor基因的重组穿梭载体rBacmid pAcP450nor。分离提取重组Bacmid DNA, 并转染培养的昆虫细胞Sf9, 得到重组病毒rAcp450nor。经酶切和PCR 鉴定, 细胞色素P450nor基因正确地插入到病毒基因组的多角体蛋白基因启动子下, SDSPAGE分析证明：表达蛋白的分子量为43kD左右。Western blotting分析结果表明：有一条特定的杂交带存在, 且分子量相同（约43kD）。进一步证明了含有真菌细胞色素P450nor基因的重组表达载体和重组病毒构建成功,并在昆虫细胞Sf9中实现了高效表达, 经MTT法测定表达的细胞色素P450nor具有还原NO的生物学活性。     

Fast and efficient generation of recombinant baculoviruses by in vitro transposition

A novel recombinant bacmid, bEasyBac, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBac, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBac bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBac was transposed with pDualBac-EGFP, the resulting recombinant virus, AcEasy-EGFP, showed comparable levels of EGFP expression efficiency to the plaque-purified recombinant virus AcEGFP, which was constructed using the bAcGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified AcEasy-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.     

A novel host-vector system for direct selection of recombinant baculoviruses (bacmids) in Escherichia coli

Site-specific transposition in Escherichia coli was used to introduce foreign genes into the Autographica californica nuclear polyhedrosis baculovirus genome. Using a temperature-sensitive donor plasmid and an E. coli host strain with an occupied Tn7 attachment site it was possible to select directly for ‘bacmid’ recombinants at 44°C. A blue to white color screen provided further confirmation of insertion at the correct site in the baculovirus genome. After cloning the gene of interest into a donor plasmid, a single transformation and plating on selective medium resulted in homogeneous baculovirus DNA which could immediately be transfected into insect cells. The utility of the host-vector system for expression in insect cells was illustrated using three heterologous genes encoding β-glucuronidase, human N-myristoyl transferase and murine preproguanylin. Using this approach, bacmid recombinants could be produced at a frequency of 10⁵ per pg input DNA. This system should not only greatly enhance the ability to obtain recombinant viruses for heterologous protein production, but should also be useful for protein engineering applications and expression cloning in insect cells.     

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.     

禽呼肠孤病毒σC基因在杆状病毒表达系统中的表达及其活性鉴定

本研究利用Bac-To-Bac杆状病毒表达系统构建重组禽呼肠孤病毒(Avian reovirus,ARV)σC基因的杆状病毒,感染sf9细胞获得表达重组蛋白。首先,将ARVσC基因克隆至pFastBacHTA载体,构建重组供体载体pFσC,将其转化大肠杆菌DHl0Bac感受态细胞,使σC基因整合到Bacmid穿梭载体中,获得重组穿梭载体BacmidσC。通过脂质体介导将其转染到sf9昆虫细胞中,获得重组杆状病毒rBacσC。通过Western Blot、间接免疫荧光试验(IFA)进行检测,结果显示:σC蛋白在重组杆状病毒rBacσC感染的sf9昆虫细胞中获得正确表达,分子质量约为37kD,表达的σC蛋白具有良好的反应活性。     

Use of transposons for studying the genetic organization of Vibrio cholerae non 01]

The nonagglutinating vibrions having Tn-elements inserted into the chromosome were obtained as a result of conjugal transfer of vector plasmids carrying the different transposons (Tn9, Tn10, Tn601, Tn5-Mob) and of the consequent isolation of plasmid-free clones of Vibrio cholerae non OI. Identification of auxotrophic mutations induced by the transposons inserted into the bacterial genome made possible the construction of the primary chromosomal map of Vibrio cholerae non OI. The efficient donor strains of Vibrio cholerae non OI were constructed by introducing the transposon Tn5-Mob and the helper plasmid RP-4. The donors are capable of oriented conjugal transfer of chromosome.     

蛋白激酶D1催化结构域在昆虫细胞/杆状病毒表达系统内的表达、纯化和活性测定

蛋白激酶D(Protein kinase D,PKD)是一种新的丝氨酸/苏氨酸蛋白激酶家族和甘油二酯(Diacylglycerol,DAG)受体,参与细胞内多种生理生化过程。为获得高纯度的PKD1的催化结构域(PKD1-cat)用于晶体学结构的研究,将带有GST标签的PKD1-cat基因克隆到杆状病毒转移载体pFastBac1中,构建了重组质粒。将重组质粒转化到含穿梭载体Bacmid的DH10Bac感受态细胞中,转座后获得了含目的基因GST-PKD1-cat的重组Bacmid。重组Bacmid DNA转染Sf9昆虫细胞后,获得重组杆状病毒并扩毒。将毒种以5 PFU/cell的感染复数感染悬浮培养的T.ni昆虫细胞,SDS-PAGE和Western blotting检测表达产物。结果显示,表达产物在分子量约68 kDa处有一特异条带可与GST单克隆抗体发生反应。经谷胱甘肽琼脂糖凝胶亲和层析纯化和PreScission Protease切除GST标签后,得到了纯度很高的分子量约42 kDa的目的蛋白PKD1-cat。体外PKD激酶活性实验结果显示,随着PKD1-cat浓度的增加,激酶活性增高。这些结果显示截短的重组PKD1-cat有很高的催化活性和纯度,为采用核磁共振或晶体学方法解析PKD1-cat的三维结构奠定了基础。     

Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase

The piggyBac Lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured TN-368 cells. We have constructed genetically tagged piggyBac elements that permit analysis of excision when transiently introduced on plasmids into the piggyBac-deficient Spodoptera frugiperda IPLB-SF21AE cell line. Precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumably supplies the piggyBac transposase. The results suggest that the piggyBac transposon encodes a protein that functions to facilitate not only insertion, but precise excision as well. This is the first demonstration of piggyBac mobility from plasmid sources in uninfected Lepidopteran cells.     

Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the γ subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Co^r, Zn^r, and Cd^r), Alcaligenes eutrophus (a member of the β subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.     

人视黄醇结合蛋白4在杆状病毒系统中的表达及其多克隆抗体制备

旨在利用杆状病毒系统表达、制备人视黄醇结合蛋白(RBP4)并检测其免疫原性。将人RBP4基因片段及信号肽SS64片段亚克隆到杆状病毒转移载体pFastBac-dual(pFBd)中,获得相应的重组转移质粒;转化大肠杆菌菌株DH10bac,转座后经筛选获得重组穿梭质粒rbacmid,将重组穿梭质粒转染孔板培养的Sf9细胞,获得含人RBP4表达框的重组杆状病毒,经过扩增获得毒种。毒种感染对数生长期的Sf9细胞并表达人RBP4蛋白(I-RBP4),通过SDS-PAGE和Western blotting对表达蛋白进行检测和鉴定。用毒种感染悬浮培养的Sf9细胞制备一批RBP4蛋白,完成SDS、Western blotting的检测及少量的多抗制备。纯化重组蛋白并与E.coli重组人RBP4(E-RBP4)分别免疫家兔。实验结果,酶切鉴定及测序证实重组转移质粒构建正确;成功构建重组RBP4-bacmid;人RBP4蛋白在昆虫细胞获得高效表达。表达的RBP4蛋白可以分泌到培养基中,分子量约为23 kDa,经过计算表达量为100 mg/L;纯化蛋白免疫兔子制备了多抗血清,血清滴度为1∶100 000,高于原核表达的抗体滴度(1∶10 000),与人体提纯蛋白制备的抗体滴度相近。杆状病毒系统高效表达了人的RBP4蛋白,具有较好的抗原性,并获得高亲和力的抗血清,为下一步的人血RBP4检测试剂盒的制备打下了坚实的基础。     

Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production

In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.     

纤维连接蛋白细胞结合区功能多肽在杆状病毒表达系统中的表达与纯化

目的：利用Bac-to-Bac杆状病毒表达系统表达纤维连接蛋白（FN）细胞结合区功能多肽（CBD）,并对其进行纯化和鉴定。方法：经PCR获得人血浆FN-CBD基因,酶切后定向克隆到T载体上,经测序正确后插入pFastBacHTB载体,转化大肠杆菌DH10Bac感受态细胞;用抗生素平板筛选重组杆粒,脂质体介导重组杆粒转染sf9昆虫细胞并进行蛋白表达;经Ni-NTA层析柱对重组多肽进行纯化,对纯化的多肽行SDS-PAGE和Western-blot分析。结果：得到融合6个组氨酸残基的FN-CBD,SDS-PAGE显示其相对分子质量约为36000,Western-blot表明该多肽能与FN的多克隆抗体结合。结论：利用Bac-to-Bac杆状病毒表达系统能成功表达出人血浆FN-CBD,且表达产物具有良好的免疫原性,为后续结构、功能研究奠定了基础。     

piggyBac转座子AgoPLE1.1在黑腹果蝇种系转化中的应用

[目的]通过检测黑腹果蝇 DDrosophiila melanogaster中piggyBac(PB)转座子AgoPLE1.1的转化活性,明确AgoPLE1.1开发为昆虫转基因载体的潜力.[方法]构建AgoPLE1.1转座酶辅助质粒pAgoHsp和带有红色荧光标记的供体质粒pXLAgo-PUbDsRed,辅助质粒和供体...     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production

The plasmid responsible for agrocin 84 biosynthesis by Agrobacterium radiobacter strain K84 has been genetically isolated free from any opine-catabolic plasmids. This was accomplished by mobilizing the agrocin plasmid, pAgK84, into a Ti plasmid-free A. tumefaciens strain, A136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37 °C. Derivatives of strain A136 harboring both plasmids or pAgK84 only produce agrocin 84. The agrocin plasmid isolated from these strains is indistinguishable by restriction endonuclease analysis from that in strain K84. A physical map of pAgK84 has been constructed with respect to six restriction endonucleases. The plasmid is cut only once by XbaI and twice by HpaI. Hybridization analysis shows that pAgK84 is closely related to pAtBo542a, a 25-Mdal plasmid from a virulent, agrocinogenic A. tumefaciens strain of European origin. Similar analyses indicate, however, that pAgK84 shows no detectable homology to octopine or nopaline-type Agrobacterium plasmids.     

杆状病毒表达系统的改进及IL-6在昆虫细胞内的表达和纯化

使用同源重组方法,在昆虫细胞内将多角体启动子驱动的EGFP表达盒插入杆状病毒穿梭载体Bacmid的p74位相,经5轮空斑纯化获得重组穿梭载体Bacmid-egfp。然后将Bacmid-egfp转化含转座助手质粒的E.coliDH10B,获得受体菌E.coliDH10Bac-egfp,由于Bacmid-egfp保留了完整的转座结构和α互补功能,因此该菌株和原始E.coliDH10Bac一样能有效的利用各种pFastBac系列的载体进行转座并构建出能指示病毒繁殖和目的基因表达的重组病毒。使用红色荧光蛋白DsRed对系统进行了验证,结果表明重组病毒Bac-egfp-DsRed感染的细胞中绿色荧光蛋白和红色荧光蛋白均得到了高效表达。进一步使用该系统在昆虫细胞中高效表达并纯化了IL-6蛋白,为研究和应用该细胞因子提供物质基础,同时也进一步证明所改造的杆状病毒表达系统的可靠性和实用性。     

汉坦病毒H8205株G1P基因片段重组杆状病毒表达载体的构建及表达

将汉坦病毒H8205株G1P基因的保守序列(约1000bp)作为目的基因插入到BactoBac杆状病毒表达系统的pFastBacHTb供体质粒中,利用Tn7转座子同BacmidDNA同源重组,获得了含目的基因片段的重组杆状病毒DNA,并利用其转染Sf9昆虫细胞,72h后收集细胞悬液,再用该悬液侵染Sf9昆虫细胞,48h后收获病毒.采用IFA分析收获的产物,观察到了特异性的荧光,并且采用SDSPAGE和Western印迹也获得了与预期一致的结果.证明感染后的Sf9昆虫细胞所表达的蛋白中含有能与抗汉坦病毒H8205株多克隆抗体特异性结合目的蛋白.研究表明,采用杆状病毒表达系统可以成功表达出汉坦病毒H8205株包膜糖蛋白G1基因片段,为开发适合的以G1P为抗原的汉坦病毒诊断试剂进行了前期的探索.     

Curing the Megaplasmid pTT27 from Thermus thermophilus HB27 and Maintaining Exogenous Plasmids in the Plasmid-Free Strain

Stepwise deletions in the only plasmid in Thermus thermophilus HB27, megaplasmid pTT27, showed that two distantly located loci were important for maintenance of the plasmid. One is a minimum replicon including one gene, repT, coding a replication initiator, and the other encodes subunits of class I ribonucleotide reductase (RNR) for deoxynucleoside triphosphate (dNTP) synthesis. Since the initiator protein, RepT, bound to direct repeats downstream from its own gene, it was speculated that a more-downstream A+T-rich region, which was critical for replication ability, could be unwound for replication initiation. On the other hand, the class I RNR is not necessarily essential for cell growth, as evidenced by the generation of the plasmid-free strain by the loss of pTT27. However, the plasmid-free strain culture has fewer viable cells than the wild-type culture, probably due to a dNTP pool imbalance in the cell. This is because of the introduction of the class I RNR genes or the supplementation of 5′-deoxyadenosylcobalamin, which stimulated class II RNR encoded in the chromosome, resolved the decrease in the number of viable cells in the plasmid-free strain. Likewise, these treatments dramatically enhanced the efficiency of transformation by exogenous plasmids and the stability of the plasmids in the strain. Therefore, the class I RNR would enable the stable maintenance of plasmids, including pTT27, as a result of genome replication normalized by reversing the dNTP pool imbalance. The generation of this plasmid-free strain with great natural competence and its analysis in regard to exogenous plasmid maintenance will expand the availability of HB27 for thermophilic cell factories.