2003 Vol.18(5)

Brief Reports

Expression of vp8 Gene of Rotavirus A in Prokaryotic Expression System

HAO Yong—hua, KUANG Zhi—zhou, XU Yang

2003, 18(5): 411

A cDNA fragment of the vp8 gene from Rotavirus strain Wa was obtained by RT-PCR and cloned into the expressing vector pGEX-5x-1 to construct recombinant plasmid pGEX-VP8. Transformation of pGEX-VP8 to E.coli JM109 and positive clonse was select, The fragment vp8 was sequenced. With IPTG inducing, the recombimant productivity was detected by SDS-PAGE. The results demonstrated that the sequence of vp8 was correct and the productivity of VP8 recombinant protein was peaked after IPTG induction in 6-8h.

Cloning and Expression of the Nonstructural Protein(NS 1)of the H9N2 Chicken Influenza Virus

LIU Jin—hua, WU Qing—min, SHI Wei—min, GUO Yu—pu

2003, 18(5): 503

Abstract:The nostructural protein(NS 1)encoded by gene 8 of the Influenza virus is present in cells infected with Influenza virus.In this study,NS 1 protein gene of the Chicken influenza virus A/chicken/Beijing/2/97(H9N2)strain was amplified by PCR.The fragment contains EcoR I and Xho I restriction enzyme sites at the ends.The amplified product was cloned into the expression vector pET-30(c).Recombinant plasmid pET/NS 1 was transformed into E.coli BL2 1(DE3)competent cells an d induced with 0.4 mmol/L IPTG the target protein was produced.the molecular weight of the expressed protein was 30 kDa as expected.Western—blot test indicated that the expressed protein Can react with the NS 1 monoclonal antibody of the influenza virus.This study laid an importan t foundation for H9N2 subtype avian influenza surveillance in China.

SOD Enhances Infectivity of Heficoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Against H.armigra Larvae

ZHOU Ming—zhe, SUN Xin—cheng, HU Zhi—hong, SUN Xiu—lian

2003, 18(5): 506

Abstract:Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV)has been used as a com m ercial pesticide for the control of cotton bollworm .H owever, some kinds of phenolase (particularly peroxidase)secreted by cotton glands,which generate free radicals,diminish the infectivity of baculoviruses significantly on cotton leaves.Superoxide dismutase(SOD)acts on the polyphenolic substance an d eliminates free radicals.Results in this research indicated that SOD—additive HaSNPV has a signi fican tly higher efi cacy to kill the bollworm on cotton leaves than HaSNPV alone.Therefore, SOD can be a promi sing enhan cer for the HaSNPV pesticide.
Research Article

Expression of Hantaan virus S Gene in B Lymphoblastoid Cell Lines from Patients with Hemorrhagic Fever with Renal Syndrome

WANG Ping—zhong, BAI Xue—Fan料, PAN Lei, ZHANG Ying, HUANG Chang—xing

2003, 18(5): 415

Peripheral blood mononuclear cells(PBMCs)were separated on Ficoll—Hypaque density gradient centrifugation from patients with Hemorrhagic Fever with Renal Syndrome (HFRS)and inf~ted by EB virus(EBV),in Order to establish immortalized B lymphoblastoid cell line(B—LCL). B—LCLs were then infected with the recombinant vaccinia viruse harboring Hantaan virus fHTNV)S gene.Expression of nucleocapsid protein was detected using indirect immunofluorescent assay(IFA). The results showed th at imm ortalized B—LCLs were set up.after B lymphocytes were infected by EBV an d cultured for 4 weeks.B lym phocytes tran sform ed by EBV becam e enlar ged an d further gath ered together.The HTNV nucleocapsid protein can be efectively expressed in B—LCLs仃ansinfected with recom—binant vaccinia viruse harboring HTNV-S gene.

Expression of Hantaan virus S Gene in B Lymphoblastoid Cell Lines from Patients with Hemorrhagic Fever with Renal Syndrome

WANG Ping—zhong, BAI Xue—Fan, PAN Lei, ZHANG Ying, HUANG Chang—xing

2003, 18(5): 420

Abstract:In order to construct the eukaryotic expression vector of月“man immunodeficieacy virus 1 (HIV-I)core protein gene(pCI—neoGAG),gag gene was acquired from the plasmid pKSGAG by restriction endonuclease digestion (Xba I ISal I)and ligated onto the eukaryotic expression vector pCI.neo.The construction of pCI.neoGAG was confirmed by restriction endonuclease digestion(Xba I ISal I)analysis and DNA sequencing.Th en the pCI-neoGAG was transfected into p8 1 5 cells by lipofectine and the expressed product was detected by indirect immunofluorescence after G4 1 8 selection for 4 weeks.The results showed that the core protein was expressed in p8 1 5 cells successfully.which will be used for the further test of HIV.1 DNA vaccine.

Replication and Expression of HCV la/lb Chim eric Genome in Transfected HepG2 Cells

WANG Hong—wei, ZHAO Ping, XIE Nan, ZHANG Min, ZHU Shi—ying, QI Zhong—tian

2003, 18(5): 423

Abstract:T0 investigate the replication and expression of Hepatitis C virus(HCV)1 a and lb chimeric cDNA in HepG2 cells.HCV l a/ l b chimeric CDNA was used to construct an expression plasmid for the tran sfection of HepG2 cells.HCV protein and HCV genomic RNA an d an ti—genomic RNA in the tran sfected HepG2 cells an d the culture supernatan ts were detected by immunocytochemical staining, W estern blotting an d RT.PCR respectively.T|le results showed that HCV NS3 protein with about 70kI)a was detectable in the tran sfected HepG2 cells.HCV genomic RNA and an ti—genomic RNA were found positive both in the HepG2 cells an d the culture supernatan ts for more than 20 generations.HCV la/lb chimera Can replicate an d express in HepG2 cells.suggesting that the RNA—dependent RNA polymerase (RdRp)of HCV lb Can initiate the replication of HCV containing genotype la untran slated region(UTR). The alterations of 5 UTR of HCV lb at nucleotides ll,12,l 3,34 and 35 dO not influence its binding to ribo some.The 3 UTR at nucleotides 9400,9403 and 9407,the deletion ‘ at nucleotide 9439 an d insertions ‘1fr’an d ‘AAT’at nucleotides 9409,9410 and 9495,9496,9497,do not significan tly influence the RdRp binding an d activities of HCV lb.ThiS HCV chimeric cDNA Can be Of value in the studies of HCV replication an d expression.

Construction of Prokaryotic Expression Vectors Harboring HV S and S Partial Gene Segments and Identification of Expressed Nucleocapsid Protein

LI Guang—yu, HUANG Chang—xing, PAN Lei, MOU Dan —lei, LI Xing—hong, ZAN G Yin, YANG Wei—song, BAI Xue—fan

2003, 18(5): 428

Abstract:To study the antigenicity of prokaryotic expressed products of Hantavirus(HV)S partial gene(502—1326bp)an d obtain data for serotyping antigen,the prokaryotic expression vectors pRSET A—S,pRSET A—S—C were constructed respectively.The recombinant expression plasmids were fans— formed into BL2 I(DE3)pLySs.Th e expression product were analyzed by SDS—PAGE and Western—blot. Th e results showed that the proteins were expressed at high level respectively an d could be recognized by patients’(infected by HTNV)sera distinctly and peculiarly.Th e truncated NP(aa155 to 429)could be considered as serotyping an tigen.

Structural Protein rp 7 Gene Sequence Analysis of Human Group B Rotavirus Strain W H-2

TANG Shao, TANG Li, WANG Bin, Nobumichi Kobayashi, YANG Ji

2003, 18(5): 432

Abstract:The VP7 gene(vp7)ofHuman group B rotavirus(GBRV)strain WH一2 was amplified and cloned into the plasmid pUCm—T by reverse transcription polymerase chain reaction(RT-PCR).Positive vp7 gene cloned plasmid pT_VP7 was sequenced and analyzed by software GeneBee,the nucleotide sequence of WH一2 vp7 gene showed high identities(more than 9O%)to that of other human GBRV,with low identities(1ess than 65%)to that of animal GBRV.The secondary structure of WH-2 vp7 mRNA was constructed by software RNAstructure 3.7 an d RnaViz 2.0,18 stem—loop structures were found on it. Th e polypeptide encoded by W H一2 vp7 gene contains 249 amino acids with a calculated molecular weight of 28.4 kDa.The protein contains three potential N—linked glycosylation sites an d a hydrophobic signal—liked sequence at its amino termina1.Comparied with other GBRV,WH一2 vp7 demonstrated 99% an d 95% of am ino acids similarity with ADRV and CAL.1 respectively,only 5 1% witIl IDⅡ .Th ese findings suggested that W H一2 and ADRV might originate from a common an cestral virus,district from an imal W H一2 vp7 reported SO far.

RT-PCR M ethods for Amplifying M Segment of Phleboviruses

LIU Dong—ying, XIAO Hong, GUO Guang—song, WEN Li, YE Meng—yi, YANG Zhan—qiu

2003, 18(5): 437

To amplify M fragment from unknown Phleboviruses,members of 7 serocomplexes and 8 no complex assigned Phleboviruses,total 42,were chosen and tested.Tl1e M segment amino acid sequences Of 4 Phleboviruses in GenBank were aligned.Conserved regions were selected to design primers. OligOnucleOtides were synthesized according to the cDNA sequences at the conservative regions.Then the OligOnucleOtides of the same region were mixed together as“cocktail"primers for RT-PCR.The amplification products were exam ined by agarose gel electrophoresis,purified an d sequenced directly. Th e am plification ratio with primer pair Ph—M一2FM and Ph—M一3RM was 8 1.0% (34,42),the size of the products were around 600bp.Tl1e am plification ratio with Ph—M一2FM an d Ph—M 一4R2M was 52.3% (22/42),the size of the products were around 1 400bp.BLAST search showed that the sequences of am plicons were homologous with known sequences of Phleboviruses.In this study,partial M fragment of sequence unknown Phleboviruses were amplified an d sequenced.TIle methods described here will be useful for genetic determination,phylogenetic analyses of Phleboviruses,an d diagnosis of Phlebovirus infections.

Construction of Recombinant Vaccinia Virus for Expressing gagpol Gene of Chinese Epidemic HIV-I Strain(CN54,clade B’/C)

National Centerfor, AIDS Control, and Prevention, National Key, Laboratory ofAgricultural, Life Science, and Technology, Huazhong Agriculture

2003, 18(5): 441

Abstract:To develop live—vectored vaccine of Human immunodeficiency virus (HIV-I)without selectable marker,we first constructed a tran sfer plasmid pVI75 with selectable m arkers of neo an d/acZ gene,an d a recombinant plasmid pVI75一Gagpol containing the gagpol gene of Chinese predominan t prevalent HIV-I strain CN54,pE/L upstream as the promoter.CEF was tran sfected by the recombinan t plasmid pVI75一Gagpol,one hour after being infected with Tiantan vaccinia virus.A recombinan t vaccinia virus rVV-Gagpol without selectable marker was constructed through two homologous recombinations as following:first,through three cycles of plaque purification under G4 1 8 pressure,the blue recombinan t virus including both ga~ ol gene and the selectable marker gene was acquired;then through three cycles of plaque purification without G4 1 8 pressure,the white recombinan t virus with ga~ ol gene but without the selectable marker gene was acquired.Thus,a recombinan t vaccinia virus was acquired.PCR an d Dot blot assay showed that the recombinant rVV-Gagpol lost the neo gene and lacZ gene.Gagpol gene could be detected by PCR.Antibody staining an d Western blot results indicated this recombinant vaccinia virus could successfully express HIV Gagpol protein.

Expression,Purification and Biological Activity Study of Viral Chemokine VM IP2

M A Xin—feng。, ZHANG Zhi—ping。, DU Tao。, YANG Ji—hong, HU Qin—xue。

2003, 18(5): 446

Abstract:ChemokiRe receptor CCR5 and CXCR4 are principal coreceptors for HIV Blockage of Human immunodeficiecy virus(HIV)infection can be achieved by engaging CCR5 and CXCR4 with their natural ligands. man herpesvirus 8 encodes three chemokines: vM IP1.vMIP2 and vM IP3.vM Ⅱ’2 has been shown to bind a range of receptors including CCR5 and CXCR4.In this study,we report the expression and purification of recombinant vM IP2 from Escherichia coli.vM IP2 gene was cloned into pET-32a(+)expression vector,which allows production of the desired protein along with a thioredoxin fusion tag.The vector containing the sequence encoding the mature form of vM IP2 was transform ed into AD494(DE3).After induction.TrxA—VMIP2 fusion protein was purified using Ni chelating column. Cleavage of the thioredoxin fusion tag was subsequently carried out with enterokinase.The cleaved protein was further purified by cation exchange column.W estern blotting indicated that purified V~皿2 had specific immunological activity with vM IP2 antibody./n vitro infection demonstrated that vM Ⅱ’2 potently inhibited the replication of R5 and X4 HIV in human peripheral blood mononuclear cell (PBMC).This study provides basis for development of efective prevention strategies against HIV Further investigation may help to define the role of vM IP2 ifl HHV8 pathogenesis.

Cloning and Expression of Nucleocapsid Protein Gene of SARS Associated Coronavirus

ZHU Han—ping, LU Qun—ying, LU Yi—yu, YAO Ping—ping, XU Fang, GE Qong, WENG Jing—qing, YAN Ju—ying, GONG Li—ming, SHI W en, ZHAO Zhi—ya, ZHU Zhi—yong

2003, 18(5): 451

Abstract:Nucleocapsid gene of SARS·-associated coronavirus(SARS·-CoV)was obtained by reverse tran scription and polymerase chain reaction from a pateint sufered from severe acute respiratory syndrome(SARS)from Beijing,subsequently cloned into pUCm—T vector.The sequence of positive recombinants was determined by the method of dideoxy chain termination,which revealed that the nucleocapsid gene segment is 1 269 nucleotide in length with an open reading frame encoding a protein of 422 am ino acids.Nucleocapsid gene was subcloned into the prokaryotic vector pET28a.Th e recombinant plasmid pET28a—SN was transformed into host cell BL21(DE3).After inducing by IPTG~ about 50kD protein was expressed and the expression level was about 45% .W estern—blot an alysis demonstrated that the recombinant proteins reacted with SARS positive sera but not with norm al sera tested. SARS nucleocapsid protein expressed by the E.coli system ofer a safe source of specific antigen for diagnostic purposes.

YANG Yuying。 , YIE Jianqiang ,ZHAO Zhen—hua。 , QIN Ai-jian ,GU Yufang

YANG Yu.ying。, YIE Jian.qiang, ZHAO Zhen—hua。, QIN Ai-jian, GU Yu.fang。

2003, 18(5): 454

Abstract:A strain of Avian leukosis virus(ALV.J1 subgroup J was isolated from an adult broiler breeder folck.which had a history of myeloid leukosis(ML).in Inner Mongolian.Moreover,another strain of ALV J was isolated from a ML case in Shandong.The two isolates were positive reaction in polymerase chain reactions with two pairs of primers specific for ALV-J and gave antigenically strong reaction in the indirect fluorescence antibody assay(IFA)with ALV-J specific monoclonal antibody JE9. The sequences of the 2.2kb PCR products of the tWO strains had 96.9% homology,an d had 94.2% — 95.4% homology to strain SD9901 and YZ9901 AL\LJ.

Rapid Selection of the Cells Persistently Infected with Foot-and-mouth disease virus and the Study of Natures in Persistent Infection Cells

GU Chao-jiang, ZHENG Cong—yi, WU Ji—bin, QU San—fu, YANG Wei, CHANG Hui—yun

2003, 18(5): 459

Abstract:A baby hamster kidney cell line(BHK一21) infected by foot—and-mouth disease virus (OGBF1 5 strain)was treated with NH4Cl,then the cells survived were selected and cloned.32 positive—cloned cell strains were obtained.One strain fBHK—ROp)was randomly selected and subcul— tured as usual and an alysed with RT_PCR.transmission electron microscope an d flow cytometry.The results demonstrated that the 4th,1 6th,36th generation BHK—ROp cell still harboured ⅥDV which had no chan ges on the growth features of the cells.They had the radical characteristics of viral persistent infection.As noted above,the NH4Cl weak base method used for establishment of the cell line persistently infected with virus was particularly effective.

Partial Characteristic Comparison Between Two Aquareovirus Isolates

FANG Qin, DING Qing—quan, WANG Ya-ping, ZHU Zuo—yan

2003, 18(5): 464

Aquareoviruses,which belong to a newly identified genus of the family Reoviridae,are agents infecting all kind of aquatic animals.Grass carp reovirus(GCRV)was isolated from outbreak grass carp fingerling in South China freshwater region,and Threadfin reovirus(1f1W )is a Singapore isolate from acute diseased marine threadfin fingerling.In this study,the partial characterization between GCRV and TFV were compared.It showed that two viruses Can cause CPE in CIK cell line,but not all the same in the other compared fish cell lines.In addition.their RNA genome compo sitions ale diferent by both electrophoresis and RT_PCR.It is interesting to point out that.based on serological related Westem blot analysis.me antiserum against GCRV could cross detecting some of the proteins found in TFV but not all of the proteins.It suggested they may share some similar epitopes.

Construction and Characterization of Recombinant CVI988 Expressing the Green Fluorescent Protein

ZHANG Xue-lian, FAN Wei-xing, ZHOU Yu-chuan, MIAO Denian, QIAN Ying-juan, CHEN Puyan

2003, 18(5): 468

The US2 gene nonessential for viral replication was amplified by polymerase chain reaction(PCR)with DNA from CEF cells infected by MDV vaccine strain CV1988 as template,and was cloned into T-easy vector;Th e expression cassette including GFP gene controlled by CM V promoter and enhancer was cloned into US2 gene to give rise to a tran sfer vector pGUS2GFP,then the complex of pGUS2GFP and DOTAP was transfected into CEF cells infected CVI988 strain virus.Recombinan t rCVIGFP expressing the green fluorescence protein was selected an d purified.Th e characterization of recombinant virus was evaluated n vivo and in vitro.Th e growth curve of recombinan t virus having EGFP gene are similar to that of the parent viru s in CEF cells.Th e recombinat virus expressing GFP could infect chicken and be reisolated from the chickens following intra-abdominal inoculation of rCVIGFP

Study on Immunity of Recombinant Vaccinia Viruses Containing env Gene of EIAV

DAI Chunming, ZHANG Xiaoyan, SHAO Yiming, SHEN Rongxian

2003, 18(5): 473

In order to develop new vaccine candidate of Equine infectious anema virus(EIAV),env gene of EIAV Chinese donkey leukocyte attenuated strain and its parental virus strain was inserted into the downstream of pE/L promoter of pSC65 vector,separately.The resulted recombinant vaccinia viruses were screened out by homologous recombination in TK region and the positive clone was confirmed with blue plaque assay.Protein expression was examined by W estern blot.It was shown that the recombinant vaccinia viruses successfully expressed the complete EIAV Env proteins an d induced significan t cellular an d humoral immune responses in the Balb/c mi ce by intramuscular inoculation. CTL specific lysis is 28% at most.Th e study will provide importan t elements for development of new EIAV genetic engineering vaccine.

Preliminary Study on the Enhancing Factor of Oedaleus asiaticus Entomopoxvirus

LI Yong—dan, ZHAO Chao—yang, WANG Li—ying

2003, 18(5): 478

Abstract:Oedaleus asiaticus entomopoxvirus(DaEPV) contains a factor which enhances the infection of Metarhizium flaroviride in the grasshopper Oedaleus infernalis.When the spheroids of OaEPV were dissolved with 2×SDS spheroid alkaline solution and centrifuged,the enhancing activity of the supernatant was 2.5 times higher than in the pellet.The enhancing activity of spheroid proteins depended on the alkali used to dissolve the spheroids.The greatest activity was detected with spheroid alkaline solution,followed with 0.02 mol/L NaOH,and none with 8 mol/L urea.Spheroid proteins were fractionated on a sephadex G一200 column.Two fractions were obtained an d the second fraction had the enhancing activity.SDS—PAGE indicated the molecular weight of the active fraction was40 kDa.

Expression of HaSNPV sod gene in E.coli

GAO Guo—hui, ZHANG Chuan—XI*

2003, 18(5): 482

Abstract:The coding region of superoxide dismutase was amplified by using PCR method from Helicoverpa armigera single enveloped nucleocapsid nucleopolyhedrovirus(HaSNPV)C 1 genome.The PCR product was cloned into pGEM —T-easy vector and sequenced.Th e cloned coding region of HaSNPV SOD was inserted into the expression vector pET blue一2 to form the recombinant plasmid pETblue2/HaSNPV SOD and was then transformed into E.coli DE3 for expression.The SDS—PAGE an alysis revealed that the expressed recombinan t SOD accumulated up to 37% of the total bacterial 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.

Studies on Reclassifying of Periplaneta fuliginosa Densovirus

LI Li, GUO Haitao, ZHANG Jiamin, HU Yuanyang

2003, 18(5): 486

Abstract:Periplaneta fuliginosa densovirus(pfDNV)was found in China at the first time,has been classified into the subfamily Densovirinae in Parvoviridae.However,Which genus that it be longs to has not yet been determined and is the focus of the present work.Recent advances in the investigation of pfDNV include two aspects:the sequence of the complete genome of the virus an d the threedimensional reconstruction of the virus at the resolution of 23A using the techniques of cryo—electron microscopy and image reconstruction.W ith these data.pfDNV is extensively compared in the present WOrk with other Parvoviruses with respect to the characteristics 0f the genome organisations an d three-dimensional structures.Our results do not support the current classification of pfDNV.Therefore, a suggestion is m ade to reclassify pfDNV.

Studies on Application Techniques of EoNPV Preparations

YIN Kun—shan, CHEN Hua—cai, TANG Mei-jun, GUO Hua—wai, XIAO Qiang, YAO Hui—ming

2003, 18(5): 492

Abstract:Ectropis obliqua nucleopolyhedrovirus(EoNPV)infects tea looper Ectropis obIiqua speci— tically.The application techniques of EoNPV preparations were studied in this paper.It was suggested that the optimum applied periods were 1~ 2 instar larvae of the 1st,the 2nd,the 5th and the 6th generation.The control efect was over 98% with the usage dose of 75 x 10 150 x 10 PIB/ha. Picking out spraying or surface spraying was more economical,although the amount of dilution water, sprayers,spraying methods did not affect the control efect significantly.

Expression of Non—structural Protein S6 of Rice dwarf virus in E coil and Preparation of Its Antiserum

MA Zhong—liang, YANG Huai—yi, TIAN Bo*

2003, 18(5): 496

Abstract: gene of a Chinese isolate of Rice dwarf virus fRDV)was amplified by RT.PCR and then cloned into pGEM—Teasy.Sequence analysis showed that its nucleotide sequence had high identity to a Japan ese isolate of RDV an d contmned high percent of rare codons. l6 gene was expressed m inclusion body with high yield in E.coli after being subcloned into expression vector pGEX一6P—1.Immune rabbit witll S6 protein obtained the antiserum.The an tiserum having a titer of l:3000 tested by ELISA Can specifically react wim the expressed S6 protein.Our study confn-med that the an tiserum could be used to detect S6 protein in RDV-infected rice tissues.

Expression Analysis of Envelope Glycoprotein E2 Protein of Rubella virus JR23 Strain

WANG Xiao—fan, WANG Zhi—yu, XUE Yong—lei, YAO Ping

2003, 18(5): 500

To construct the recombinant plasmid containing envelope glycoprotein E2 gene of RV JR23 strain,to express E2 protein in BHl(2 l cells,and to analyze the antigenic sites of E2 protein of RV JR23 strain.E2 gene of RV JR23 strain was amplified bv I PCR.The PCR product was cloned into the expression vector pBluescript II S and transformed into E.coli JM 109.an d the recombinant plasmid was transfected into BHK21 cells with recombinant vaccinia viru s VTF一7.The expression was detected by immunohistochemistry an d indirect immunofluorescence.The recombinan t plasmid contained glycoprotein E2 gene.and expressed well in BHK2 l cells.The expressed E2 protein mainly accumulated in plasma near the nuclei.Th ere are immunological epitopes of E2 protein on RV JR23 virions.The results showed that RV JR23 E2 transient expression system was successfully constructed. This study ofers the basis for investigation of the relationship between the structure an d fu nction of RV proteins,and interaction of viru s an d host cells.

The Action 0f NS3 Protein in the Life Circulation 0f Flavividae Family


2003, 18(5): 508