The role of hepatitis B virus x gene in development of primary hepatocellular carcinoma

Primary hepatocellular carcinoma (HCC) is one of the most common cancers occurring in human, and there is strong epidemiological evidence suggesting that persistent hepatitis B virus (HBV) infection is the most important risk factor for its development. HBx gene was found to be a transactivator recently. Its continuous expression in hepatocytes may transactivate cellular genes which can play a certain role in development of HCC. The HBx gene fragment was used to construct a recombinant eukaryotic expression vector pCEP4 and introduced into HepG2 cells. The effect of HBx gene on HCC cells growth and its molecular mechanism in HCC cells regulation were investigated.     

Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However,the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins(GPs), such as vesicular stomatitis virus G protein(VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64.Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus(AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4–12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher p H conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.     

人睫状神经营养因子在大肠杆菌和毕赤酵母中的表达和生物活性测定

The recombinant human ciliary neurotrophi factor(hCNTF)expressed in E.coli aggregatedas inclusion bodies and refolding procedure was necessary to obtine the active protein.To overcome the disadvantage,we cloned hcntf gene into yeast expression plasmid pPIC9K and collected the plasmid pPIC9K-hcntf.Plasmid pPIC9K-hcntf was transformed into yeast Pichia pastoris GS115,and screened on G418-SD plates.The transformants with high copies of hcntf gene were inoculated into BMMY media and induced with 0.5% methanol.The recombinant hCNTF was secreted into the media.The amount of hCNTF in the supernatant was about 10 mg/L when incubated in the conical flasks and reached up to 60 mg/L under fed-batch condition in 15 L fermentator.The recombinant hCNTF expressed in E.coli was renatured as the control.The neonatal rat dorsal root ganglion assay showed that proteins expressed in both systems have the activity of promoting the growth of neuron axons.The phenomenon can be observed with only 3 μg hCNTF expressed in yeast present,which indicates that hCNTF was successfully expressed in Pichia pastoris and has a relatively high activity.     

铜绿蝇蜕皮激素受体在杆状病毒载体系统中的表达

The baculovirus expression vector, Trichoplusia ni nucleopolyhedrovirus, with the advantage of polyhedral inclusion body formation in recombinant viruses, was used to express the ecdysteroid receptor of the Australian sheep blowfly Lucilia cuprina(LcEcR). pSXIVVI +X3/2 baculovirus transfer vector was chosen for a 2.8kb LcEcR cDNA subcloning since pSXIVVI +X3/2 contains an efficient translational initiation signal (ATG) and it allows the LcEcR cDNA fusion to N-terminal codons in the correct reading frame. The resulting transfer plasmid pSXIVVI +X3-LcEcR was cotransfected into BT1-Tn-5B1-4 cells with the parental virus TnNPV-SVI -G minus polyhedral inclusion body, which expresses β-galactosidase gene. After 3～4 runs of plaque purification, three TnNPV-LcEcR clones were obtained with the LcEcR gene under the dual control of synthetic and XIV promoters. These three TnNPV-LcEcR clones all showed white phenotype when stained with X-gal. Western blot analysis showed 2～3 specific polypeptides with molecular weight ranging from 70～90kD. Three TnNPV-LcEcR clones expressed different level of LcEcR polypeptides in BT1-Tn-5B1-4 cells. The TnNPV-LcEcR-1 clone expressed the highest level of LcEcR polypeptides in BT1-Tn-5B1-4 cells 48～72h post infection.     

Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses

lacZa-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome （BAC） by homologous recombination in E. coli. The resulting recombinant BAC （pBeckerZF1） was confirmed by PCR and sequencing. Green fluorescent protein （GFP） gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect （CPE） observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3, vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.     

杜氏盐藻外源基因稳定表达系统的构建(英文)

A stable transformation system for the expression of foreign genes in the unicellular greenmarine alga (Dunaliella salina Teod.) was established. Using electroporation, the alga was transformed witha plasmid containing the hepatitis B surface antigen (HBsAg） gene and the chloramphenicol acetyltransferase(CAT) gene as a selectable gene. PCR and Southern blotting analysis indicated that the HBsAEgene wasintegrated into the D. salina genome. Northern dotting analysis showed that the HBsAg gene was expressedat the mRNA level. The stable expression of HBsAg protein in transformants was confirmed by HBsAgenzyme-linked immunosorbent assay (HBsAg EUSA) and Western blotting analysis. Also, PCR and Southernblotting analyses showed that the CA Tgene was integrated into the D, salina genome, and CAT EUSAindicated that CAT protein was stably expressed in the cells. The introduced HBsAg DNA and HBsAgprotein expression were stably maintained for at least 60 generations in media devoid of chloramphenicol.This is the first report of the stable expression of foreign genes in D. salina.     

High-level expression of whiG——A key gene for Streptomyces differentiation in Escherichia coli

Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.     

Recombination and Heterologous Expression of Aliophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires theintroduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins ormetabolic pathways.In order to accomplish the expression of multiple genes in a single transformationevent,we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonasreinhardtii chloroplast expression vector,resulting in papc-S.The constructed vector was then introducedinto the chloroplast of C.reinhardtii by micro-particle bombardment.Polymerase chain reaction and Southernblot analysis revealed that the two genes had integrated into the chloroplast genome.Western blot andenzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria couldbe correctly expressed in the chloroplasts of C.reinhardtii.The expressed foreign protein in transformantsaccounted for about 2%-3% of total soluble proteins.These findings pave the way to the reconstitution ofmulti-subunit proteins or metabolic pathways in transgenic C.reinhardtii chloroplasts in a single transformationevent.     

Vertical transmission of TnSNPV,TnCPV, AcMNPV,and possibly recombinant NPV in Trichoplusia ni

Four viruses were tested for vertical transmission in Trichoplusia ni: T. ni nucleopolyhedrovirus (TnSNPV), T. ni cypovirus (TnCPV), Autographa californica nucleopolyhedrovirus (AcMNPV), and AcMNPV engineered to express a scorpion toxin (AcMNPV.AaIT). Fifth instars were exposed to each virus, the survivors were reared and mated, and second-generation (F(1)) insects were examined for infection. TnSNPV was transmitted to offspring at a prevalence rate of 15.4%, TnCPV at 10.2%, and AcMNPV at 10.1%. Only one of 2484 F(1) insects was infected with AcMNPV.AaIT; this experiment was repeated, and none of 4774 insects was infected. Thus, vertical transmission is unlikely to contribute to AcMNPV.AaIT contacting non-target organisms after its field release. There was evidence that TnCPV and possibly TnSNPV were activated to overt infections by ingestion of a different virus. TnCPV, but not the NPVs, routinely infected 0.3-1.7% of non-treated insects, probably indicating that it is vertically transmitted at enzootic levels.     

Competition between Wild-Type and Recombinant Nucleopolyhedroviruses in a Greenhouse Microcosm

Wild-type Autographa californica nucleopolyhedrovirus (AcNPV or AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing a mutated juvenile hormone esterase (AcJHE.SG) were compared in their capability to produce epizootics in larvae of Trichoplusia ni infesting collards in a greenhouse microcosm. Larvae treated in four different ways were released into 1.8-m² microplots in week 1. The four treatments included (1) uninfected larvae (control), (2) 100% AcNPV.WT-infected larvae (WT), (3) 100% AcNPV.AaIT-infected larvae (AaIT), and (4) 1:1 ratio of AcNPV.WT-infected and AcNPV.AaIT-infected larvae (WT+AaIT). On a weekly basis, larvae were sampled and new, uninfected larvae were added to all plots. Sampled larvae were reared until death and then subjected individually to DNA–DNA dot-blot hybridization assay to determine the proportion of insects infected with each virus in each plot. The entire experiment was repeated with AcJHE.SG in the place of AcNPV.AaIT. Epizootics of AcNPV.WT lasted 8 weeks after a single viral release in the replicated greenhouse microplots. AcJHE.SG epizootics also lasted 8 weeks after viral release, but this virus and AcNPV.AaIT were both out-competed by AcNPV.WT. AcNPV.AaIT was no longer detected in the T. ni population by the fourth week after release. AcNPV.WT also increased to greater numbers in soil than AcNPV.AaIT or AcJHE.SG after 8 weeks. Thus, it was possible to induce 8-week epizootics of AcNPV.WT in replicated microplots under artificial greenhouse conditions, and the wild-type virus out-competed the recombinant virus for a niche in this greenhouse microcosm, which reduces the probability that the recombinant virus will persist in an agroecosystem.     

利用杆状病毒表达系统表达金鱼生长激素I基因

以不含起始密码ATG的质粒Psxivvi⁺X3为转移载体,将编码金鱼生长激素I的Cdna插入粉纹夜蛾核型多角体病毒(TnNPV)基因组中,构建了形成多角体的重组病毒株TnNPV—SX⁺gfGHl21a。该毒株能利用合成多角体XIV串联启动子,在重组病毒感染的草地贪夜蛾(Spodoptera frugiperda,Sf)昆虫细胞及银纹夜蛾幼虫中表达金鱼生长激素I基因。感染离体细胞及虫俸后的蛋白SDS聚丙烯酰胺凝胶电泳表明．所表达的蛋白分子量为22．5kDa,与理论计算值相符。Westem印迹证实。金鱼生长激素特异蛋白得到表达。     

Firefly luciferase, synthesized to very high levels in caterpillars infected with a recombinant baculovirus, can also be used as an efficient reporter enzyme in vivo

Trichoplusia ni and Spodoptera littoralis larvae were infected with a recombinant AcNPV, having the viral polyhedrin gene replaced with the cDNA encoding firefly luciferase. Both S. littoralis and T. ni synthesized very high levels of luciferase representing greater than or equal to 25% and greater than or equal to 15%, respectively of the total Coomassie blue stainable protein. Luciferase was apparently not secreted into the hemolymph but was contained within the body tissue. Expression in S. littoralis larvae suggests that luciferase can be an excellent reporter enzyme to study virus infection, dissemination and expression in different tissues, host range determination, insect physiology and also to monitor the release of recombinant virus in the environment when used as a biocide.     

Transport of Wild-Type and Recombinant Nucleopolyhedroviruses by Scavenging and Predatory Arthropods

Abstract Wild-type and recombinant nucleopolyhedroviruses (NPVs) were compared in their capability to be transported over limited distances by the predator Podisus maculiventris (Say) and scavengers Sarcophaga bullata (Parker) and Acheta domesticus (Linnaeus) in Trichoplusia ni (Hübner) larvae infesting collards in a greenhouse microcosm. Viruses tested were variants of Autographa californica (Speyer) NPV (AcNPV): wild-type virus (AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing juvenile hormone esterase (AcJHE.SG). Podisus maculiventris transported AcNPV.WT and S. bullata transported AcNPV.WT and AcNPV.AaIT. Prevalence and transport of AcNPV.WT were greater than those of AcNPV.AaIT and AcJHE.SG, regardless of whether the nontarget organism carriers were present or absent. Podisus maculiventris and S. bullata transported recombinant and wild-type NPVs at a rate of up to 62.5 cm/day, and A. domesticus transported wild-type NPV at 125 cm/day. The infected host insects, T. ni, undoubtedly contributed to viral transport in the current research. In every experiment, both the wild-type and recombinant virus spread to some degree in the plots without predators or scavengers. The relative amounts of NPVs that accumulated in soil, as indicated by bioassay mortality percentages, generally exhibited spatial patterns similar to those of T. ni mortality due to NPV on the collards plants. Thus, the predator and scavengers in the current research demonstrated some capacity to transport wild-type as well as recombinant viruses at significant rates in a greenhouse microcosm. Received: 6 December 1999; Accepted: 29 February 2000; Online Publication: 12 May 2000     

Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californica, Autographa gamma, Trichoplusia ni, Rachiplusia ou, Anagrapha falcifera, Galleria mellonella, and Heliothis virescens. Alignment and phylogenetic inference from partial nucleotide sequences of three highly conserved genes (lef-8, lef-9, and polh) indicated that 45 of 74 samples contained isolates of AcMNPV, while six samples contained isolates of Rachiplusia ou multiple nucleopolyhedrovirus strain R1 (RoMNPV-R1) and 25 samples contained isolates of the species Trichoplusia ni single nucleopolyhedrovirus (TnSNPV; Alphabaculovirus). One sample from A. californica contained a previously undescribed NPV related to alphabaculoviruses of the armyworm genus Spodoptera. Data from PCR and sequence analysis of the ie-2 gene and a region containing ORF ac86 in samples from the AcMNPV and RoMNPV clades indicated a distinct group of viruses, mostly from G. mellonella, that are characterized by an unusual ie-2 gene previously found in the strain Plutella xylostella multiple nucleopolyhedrovirus CL3 (PlxyMNPV-CL3) and a large deletion within ac86 previously described in the AcMNPV isolate 1.2 and PlxyMNPV-CL3. PCR and sequence analysis of baculovirus repeated ORF (bro) genes revealed that the bro gene ac2 was split into two separate bro genes in some samples from the AcMNPV clade. Comparison of sequences in this region suggests that ac2 was formed by a deletion that fused the two novel bro genes together. In bioassays of a selection of isolates against T. ni, significant differences were observed in the insecticidal properties of individual isolates, but no trends were observed among the AcMNPV, TnSNPV, or RoMNPV groups of isolates. This study expands on what we know about the variation of AcMNPV, AcMNPV-like and TnSNPV viruses, provides novel information on the distinct groups in which AcMNPV isolates occur, and contributes to data useful for the registration, evaluation, and improvement of AcMNPV, AcMNPV-like, and TnSNPV isolates as biological control agents.     

密码子偏性对痘苗病毒载体表达效率影响的研究

为了研究密码子偏性对痘苗病毒载体表达效率的影响,分别采用痘苗病毒及其宿主细胞的优势密码子对绿色荧光蛋白基因进行改造,利用荧光、Western blot和FCM等方法分析其在痘苗病毒载体系统的表达水平。结果显示,全部采用痘苗病毒优势密码子(富含A T)和全部采用宿主细胞优势密码子(富含G C),以及部分使用宿主细胞优势密码子的三种绿色荧光蛋白基因都能够有效表达,表达水平相近,表明痘苗病毒载体对目的基因密码子的使用具有很好宽容性。为了探讨这种宽容性的机理,分别利用在胞核内和在胞浆内转录的质粒载体对不同密码子偏性的绿色荧光蛋白基因进行表达分析。结果显示,胞核内转录目的基因的pcDNA3质粒载体能有效表达富含G C的绿色荧光蛋白基因,不能有效表达富含A T的绿色荧光蛋白基因,而胞浆内转录目的基因的pSCA质粒载体能同样有效表达上述不同密码子偏性的目的基因。这些结果表明,位于胞浆内的富含A U的转录产物能够有效表达,细胞核内生成的富含A U的转录产物可能受核膜屏障或其它核内因素影响而不能有效表达。因此,胞浆内繁殖的特性是痘苗病毒载体具有密码子宽容性的主要原因。此研究为痘苗病毒载体和常用真核表达载体的选择使用提供了重要实验依据。     

Expression of the fusion protein recombinant human granulocyte-macrophage colony stimulating factor and leukemia inhibitory factor in a baculovirus vector system

A fusion gene coding human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) cDNAs was inserted into the transfer vector pSXIVVI⁺ X3 with the control of Syn and XIV promoters. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). The fusion protein recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) could be synthesized in cells infected with recombinant virus at a level of about 23% of their total cellular protein. Activity analysis of the fusion protein in infected cells revealed that it exhibited the dual activities of GM-CSF and LIF. Western blot analysis of the expressed fusion protein in infected larvae showed that the virus-mediated fusion protein, with a molecular weight of ∼35 kDa, is confirmed with immunoreactivity. Received 02 December 1998/ Accepted in revised form 07 May 1999     

Expression of post-translational processing of preprocecropin A using a baculovirus vector.

A cDNA fragment encoding preprocecropin A was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in recombinant-infected last instar larvae of Trichoplusia ni and in diapausing pupae of Hyalophora cecropia. The identity of the recombinant product was established by electrophoresis with detection of antibacterial activity and mass spectrometry. The prepropeptide had been correctly processed including removal of signal peptide and pro-part. Biologically active and amidated cecropin A was exported to the hemolymph. The yield of recombinant protein in H. cecropia reached a level of 600 micrograms/ml hemolymph and about 70% of the material was amidated.