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2003 Vol.18(1)

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Contents

HCV E2 Prodaryotic system Gene expression Purification

ZHANG Dong—wei, LIANG Bu—feng, LI Wei-dong, QI Zi—bai, LING Shi—gan

2003, 18(1): 1

Rceomdinant plasmid pET-E2 was transformed into BL21 (DE3) competent cell, then induced the amplified culture with IPTG. There was a 34 kDa band in SDS-PAGE gel. The molecular weight of the expressed protein was comsistent to that of deduced E2 protein. Tested by western-blot, It showed that the product was HCV E2 protein. It contained a six-Histidine tag and aggregated into the inclusion body. Computer scan analysis showed the protein interested took the percentage more than 36 of the total bacterial proteins and the rate of purification was higher than 95% after purified by Ni-column. The activity of pruified E2 protein was tested by ELISA. The result showed that the 5/15 HCVpositive sera had anti-E2 antibody, and the other 5 negative HCV sera hadn’t anti-E2 antibody

The Construction and Expression of Eukaryotic Expression Vector Harboring Homo Sapiens Integrin beta 3 ORF Gene Segment

MOU Dan—lei, BAI Xue-fan, LI Guang-yu, PAN Lei, HUANG Chang—xing, YANG Wei—song

2003, 18(1): 5

To study the cellular entry of Hantavirus, we constructed dukaryotic expression vectors harboring Homo sapiens integrin β3 gene. The 2.3kb ORF region of human integrin β3 subunit cDNA was amplified from the plasmid containing cDNA of human integriri β3 by PCR. Then the PCR products with the blunt-ends were directly cloned into eukaryotic ecpression vector pcDNA 3.1/V5-His TOPO by TA Cloning. It shows that the integrin gene was inserted into the plasmid vector by restriction enzyme analysis and PCR, and the sequence is identical with the reported human integrin β3 ORF gene, then the recombinant expression plasmids were transfected into CHO cells in vitro. The transient expression products were analyzed by IFA. The results above suggested that the eukaryotic expression vectors, pcDNA3.1-β3, has been successfully constructed and the expressed protein could be detected distinctly and peculiarly. It lies a basis for the further study of hantavirus pathogenicity and biological characteristics

DNA Cloning and Sequence Analysis of A TTV Isolated in W uhan

LI Jang, YE Lin-bai”, GAO Jin-rong, WU Zheng—hui

2003, 18(1): 9

A T1 DNA fragment was obtained from W uhan patient serum by PCR,which was cloned into plasmid pMDl8—-T and analyzed by sequencing combined with computer techniques comparing withother isolated strains.The results reveal that this DNA fragment consists of 1 333 base pairs containing whole Trv ORF2 and partial ORFI coding regions, the DNA and deduced amino acid sequence are highly homogenous with those of 1TrV la type.ORF2 peptide consists of 202 amino acids. whose parts of N terminal and C term inal have comparatively high hydrophilic and strongly antigenic characters than middle part.Th e part near N term inal contains a typical tyrosine kinase phosphorylation site(RARDWPGY,38-45aa) and 3 potential protein kinase C phosphorylation sites, the structure in N terminal part is mainly仅helices:while the part near C term inal contains proline rich region and 3 consecutive N-myristoylation sites,the structure in C terminal part is mainly B turns.We infer that the ORF2 protein maybe a phospholated Protein

Studies on Genetic Immunization of the Chimeric Genes of Hantaan virus M and S Segments that Spliced by Diferent W ays

ZHANG Fang—lin, XU Zhi—kai, LUO Wen, YAN Yan, WU Xing—an, LIU Yong, BAI Wen—tao, ZHAO Qian, WANG Hai—ta

2003, 18(1): 14

Abstract:Recombinant euckaryotic expression vectors pcDNA3.1二G2S0.7 and pcDNA3.1一S0.7G2 were constructed by cloning chimeric genes containing G2 fragment of M segm ent and 0.7Kb fragment of S segment into pcDNA3.1 (+、. Then BALB/c mice were vaccinated by the two vectors. EUSA results showed that both the vectors could induce specific antibodies against NP and GP.The specific antibody titers stimulated by pcDNA3.1一G2S0.7 were obviously higher、that that of pcDNA3.1一S0.7G2. M IT re— sults showed that the stimulation indexes of splenocytes of pcDNA3.1一G2S0.7 to NP and GP were sig— nificantly higher than that of contro1.However.that of pcDNA3.1一SO.7G2 were equal to that of contro1. It is suggested that the chimeric genes of Hantaan virus containing G2 fragment of M segm ent and 0.7Kb fragment of S segm ent could directly specific anti—Hantaan virus humoral immunity and cellular immunity in BALB/c mice.Th e diferent splicing ways could afect the immunity of chimeric genes.

Bioinformatic Analysis of HTRP Gene Induced by HSV-1 Binding to the Fibroblast

LIU Long-ding, SHI Hai-jing, DONG Shao-zhong, DONG Cheng-hong, WANG Li—chun, ZHAO Gang, LI Qi-han

2003, 18(1): 18

By using bioinformatic methods, the characters of HTRP-a new unknown function gene of cellular response against the specific stimulation of HSV-1 were analyzed and predicted.The preliminary speculation on the transcription regulation region and encoding protein were undertaken.Th ese data provide information basis for the further study on the function genes in the process of cellular re— sponse to HSV一1 stimulation

HIV-1 Coreceptor Chmokine Bicistronic expression vector Transfection

ZHANG Ying, BAI Xue-fan“, HUANG Chang-xing, SUN Yong—tao, PAN Lei, LI Guang—yu, WANG Lin—XH

2003, 18(1): 23

In order to construct bicistronic expression vector of RANTES and SDF-l,the ligands of HIV-1 principal coreceptors,RANTES-KDEL were amplified from pCMV-R-K by PCR and ligased with eukaryotic expression vector pCMV-S/K. The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. Then pCMV-R-K-S-K transfection into HeLa cells was carried out by lipofectin. RAN TES and SDF-1 were showed expressed in HeLa cells by indirect immumofluo-rescence. The results show that pCMV-R-K-S-K was constructed and expressed in cell line HeLa successfully, which will contribute to gene therapy of HIV-1.

Construction of Agrotis segetum granulosis virus cDNA Library

ZHANG Wei, WANG Wei, AI Xiu-lian”, XIE Yu-qing, FEN Huai-rong

2003, 18(1): 27

Abstract:A general cDNA library of Agrotis segetum granulosis virus(ASGV)which was from infected A grotis segetum(As)l arva has been constructed.The first and second cDNA strands were synthesized by AMV reverse transcriptase from mRNA which was isolated from total RNA of infected As larva. So the general cDNA library includes AsGV’S and AS’S, Th en the library of AsGV included 108 1 positive clones were screened by spot hybridization, Th e labeled AsGV genome DNA probe With DIG kit was cut by restriction enzyme of EcoR I and Hind 11. Th e sequence of positnive clones accord with the genome of AsGV,and there are 59 coding sequence which were amplified with synthetic primer of As— GV genome according entirely with the genome of AsGV.

Establishment of a cell line from embryos of Mythimna separata and its susceptibility to M sNPV

YU Hong-chun, ZHENG Gui-ling, WANG Xiao-yun, LI Guo—xun, ZHAO Kui-jun

2003, 18(1): 31

Abstract: A new cell line,designated NEAU—Ms一9803 12,was established from 24一hr—old Mythimna separata eggs and was passaged 201 times.The cells were heterogeneous in morphology with spherical and spindle—shaped cells.Th e population doubling time of the cell line aws 47.82h in 120h at 28~C. The cell line was highly susceptible to Mythimna separata nll~leal"polyhedrosis vu’us (MsNPV).When the cells were inoculated at a multiplicity of infection (m.o.i.)of 0.08TCID50/cell with wild pas— sage MsNPV。the infection rates and occlusion bodies f OB) yield reached a value of 62.10% , 33.59PIB/infected cell at 240h postinfection(pi),respectively

Time Course of Protein Expression in the Cell Infected with M utants of AcM NPV

DING Qing-quan, FANG Qin, e Lapointe, DAI Shun-yin, ZHAO Xiao-dong, Eric B

2003, 18(1): 35

In order to compare the difierence of protein expression in A utograph cdifornica nu,cleo,7 polyhedrosis virus(AcMNPV)mutants,the spodopterafrugiperda IPLB-SF-21 Cell was infected with wild type(HR3)and mutants(ts317,ts538,ts8),and incubated at permissive and nonpermissive temperature, then determ ined the time of protein expression with antibodies against P47, P143 and other viral structure proteins of budded virus.The results indicated: 11 P47 was expressed protein in late.The cells infected with mutants could be expressed the P47 at 25℃ and not at 33oC.21 P143 was expressed protein early.The cells infected with mutants could be expressed the P143 at both temperature, but ts8 was the most weak.3)Similar levels of structural proteins of AcMNPV were detected in both wild type and ts3 1 7 at 25~C,but ts538 and ts8 were slight weak than ts3 1 7-infected cel1.Almost of BV proteins(except to GP64 and P2.4)were not expressed in cells infeeted with ts538 and ts8 at 33℃ .

M olecular Cloning Dendrolimus punctatus cytoplasmic polyhedrosis virus Segment 8 by Single Primer Amplification and Sequence Analysis

HU Jian—fan.ZHANG Jia—min, YANG Juan, DENG Xiao-jun, WANG Yang, HU Yuan-yang

2003, 18(1): 39

Dendrolimus punctatus cytoplasmic polyhedrosis virus(DpCPV)was purified from infected Dendrolimus punctattts and the genomic dsRNA segments were subsequently extracted. Segment 8 dsRNA was purified by low melting-temperat agraose.A single amino-linked modified oligonucleotide (primer 1) was ligated to either 3’end of dsRNA genome segment 8 by Using T4 RNA Ligase. Following reverse transcription, annealing and repair of cDNA strand. amplification of S8 dsRNA genome was accomplished by PCR using a single complementary oligonucleotidc(primer 2).The amplified cDNA was cloned into the pMD18-T vector.Nucleotide sequence analysis of cDNA derived from the segment 8 revealed that it consists of 1 330 nucleotides encoding putative protein of 390 amino acids with molecular weith of 44KD. Comparison of the nucleolide sequence of the segment 8 of DpCPV with that of BmCPV and LdCPV showed that nucleotides homology is 83% and 97% . respectively.

Cloning and Prokaryotic Expression of Ubiquitin Gene of Spodoptera exigua multi-nucleocapsid nucleopolyhedrovirus

NIU Guo—dong, ZHANG Xiao-xia, ZHANG Zhong-xin

2003, 18(1): 44

A ubiquitin gene of Spotoptera exigue multi-nucleopolyhedrovirus (SeMNPV) was cloned and sequenced.The result showed that the gene size was 243 bp,encoded 80 amino acids with apredicted size of 9.4kDa.To construct expressive vector for the udiquitin gene of SeMNPV.the frag. ment containing ubiquitin gene was inserted into pET-28a expressive vector.Expression of the fusion protein was induced by IPTG in E.coli BL2 1 fDE3).By changing the time induced and the content of IPTG.the fusion protein had an optional expression leve1.The fusion protein was identified by West. elXl blot with a mouse Ab anti Ubiquitin from bovine oringe.To do the further study , the specific Ab anti viral Ubiquitin was produced.In addition. the sequence of amino acids was analyzed with the software of Gendoc.Th e result displayed that ubiquitin of baculoviruses had some different amino acid substitutes in some residues and the amino acid sizes varied a lot compared with eukaryotic udiquitin. From these,we reckon that ubiqtuitins of baculovirus may have a unique way in the gene evolution

M olecular Cloning and Sequence Analysis of Dendrolimus punctatus cytoplasmic polyhedrosis virus NS5 Protein Gene

WEN Li, ZHANG Jia—min, WANG Qiong, HU Jian-fang, HU Yuan-yang

2003, 18(1): 49

For the synthesis of cDNA of Dendrolimus punctaus CPV (DpCPV)NS5 protein,the primers were designed on the basis of the RNA sequmce of BmCPV一1(I strain)segment 9.After the RT—PCR the amplified cDNA was cloned into the pMD18一T. NS5 protein gene of DpCPV was found to be 963 nucleotides in length with a single open reading frame in one strand capable of coding a predicted pro— tein of 320 residues(Mw of 35.66kDa). Comparison of the nucleotide sequences of the NS5 gene of DpCPV with those of BmCPV—I(1 strain)showed that nucleotides homology iS 86.0% , but there are only 2 1 amino acid differences between the two CPVs.

Cloning and Prokaryotic Expression of groEL Gene of Endosybiotic Bacterium from rhopalosiphum maize

LIN Lin, WU Yun—feng”, CUI Xiao-feng

2003, 18(1): 54

Cloned one virus binding protein from Rhopalosiphum maize Yangling biotype through PCR technique,using designed specific primers based on this protein partially sequence.In nucleotide and amino acid sequeneee levels. this protein was identified as GroEL protein of endosymbiotic bacterium (Buchnera sp.) of aphid.The results of nucleotides sequencing indicated that the full length of groEL genes f GeneBank accessio number iS AF3 87863) iS 1 647bp and code 548 amino acids.Th e cloned groEL genes of and Rhopalosiphum maize were ligated into pBV22 1,pET30a and pTrcHisA vector for expressing The results suggested that 63 KDa aim proteins can be espressed using pBV22 1.When pET一30a used,the expressed fusion proteins have a molecular weight of 69 KDa,and both have a higher expression quantiy

Cloning and Analysis of Partial Genome Sequence of Tobacco vein distorting virus

MO Xiao, QIN Xi, YANG Cheng, DUAN Yu-qi, WU Juan, LI Tian-fei, WU Jian-yu, CHEN Hai-ru

2003, 18(1): 58

Tobacco vein distorting virus(TVDV)is one of the casual agents of tobacco bushy top disease. It was designated a tentative member of the family Luteoviridae. Partial sequence of the genome of rIvDV was amplified by RT-PCR from total RNA of tobacco affected with tobacco bushy top disease,using universal primers of Luteoviruses and degenerate primer of Poleroviruses.The 1 654 bp sequence of rIvDV genome encoded partial putative RNA-dependent RNA polymerase. coat protein and movement protein. Molecular phylogenetic trees were constructed based on the amino acid sequence of these three proteins.indicating that rIvDV should be a definitive memb er of the family Luteoviridae.According to the higher identities of amino acids of ORFs and the similar length of intergenic spacer region of rIvDV with the member of the genus Polerovirus.we proposed that rIvDV was a new member in the genus Polerovirus.Th is is the first report on the molecular properties of TVDV.

Cloning and Analysis of Partial Genome Sequence of Tobacco vein distorting virus

MO Xiao, QIN Xi, YANG Cheng, DUAN Yu-qi, WU Juan, LI Tian-fei, WU Jian-yu, CHEN Hai-ru

2003, 18(1): 58

Tobacco vein distorting virus(TVDV)is one of the casual agents of tobacco bushy top disease. It was designated a tentative member of the family Luteoviridae. Partial sequence of the genome of rIvDV was amplified by RT-PCR from total RNA of tobacco affected with tobacco bushy top disease,using universal primers of Luteoviruses and degenerate primer of Poleroviruses.The 1 654 bp sequence of rIvDV genome encoded partial putative RNA-dependent RNA polymerase. coat protein and movement protein. Molecular phylogenetic trees were constructed based on the amino acid sequence of these three proteins.indicating that rIvDV should be a definitive memb er of the family Luteoviridae.According to the higher identities of amino acids of ORFs and the similar length of intergenic spacer region of rIvDV with the member of the genus Polerovirus.we proposed that rIvDV was a new member in the genus Polerovirus.Th is is the first report on the molecular properties of TVDV.

Characteristics of the Giycoprotein cDNA and Antigenicity of Rabies virus Vaccine Strain SRV9

YUAN Hui-jun, HU Rong-liang”, ZHANG Shou-feng, ZHANG Mao—lin, TU Chang—chun

2003, 18(1): 63

0ral vaccine candidate of Rabies virus strain SRV9 is an intermediate plaque subclone of SAD B 19 and has been used in field with good safety and immunogenicity.A pair of primers was designed according to the published sequence of rabies virus glycoprotein.Reverse transcription—polymerase chain reaction(RT—PCR)was emoloyed to amplify the full cDNAs of the SRV9 and its ancestor SAD B 19.Sequencing result has shown that the RT-PCR product was 1 588bp in length in which a 1 575bp open reading frame was included.It encodes a mature polypeptide composing of 505 amino acids.Compared to SAD B19,base transversions occurred at positions 158,575 and 931,resulting in amino acid mutations at 53, 1 92 and 3 1 1 residues in polypeptide.Compared to the vaccine strain ERA in ues.ten base differences and 7 amino acid alterations were marked.Antigenic index analyses found that the mutation in 192 amino acids resuhed in the form ation of a new potential antigenic site. As the glycoprotein of rabies virus is the only antigen inducing neutralizing antibodies,the alteration of Hi Ain 197 position may be the causes of forming a special plaque and further improvement of its safety.Keeping analyses on amino acids of the glycoprotein of rabies virus is also an important印- proach of surveillance on rabies virus strains

Analysis of the Sequence of Variable Region in vp2 of Infectiousbursal disease virus Isolated from Diferent Bird Species

WANG Yong-shan, Zhou Zong-an, DENG Xiao-zhao, ZHAO Xin-tai, HOU Ji-bo, HE Kong-wang, DING Chan, LIU Yu, GAO Jian, DIAO Zhen-yu

2003, 18(1): 68

Abstract:According to the published cDNA sequences of Infectious bursal disease virus(IBDV),a pairof primers to variable region in VP2 were designed for cDNA synthesis and PCR amplification of fourIBDV isolated from different bird species,CH,DU,GE and SP.The nucleotide sequences determinedhave been compared to each other.Th e four isolates are closely related,the homology of the four iso—lates being 97% at the nucleotied level and 98% at the amino acid leve1.Th eir two hydrophilic regionsand one heptapeptide were identical at the amino acid leve1. Th e results revealed that the ducks,geeseand sparrows naturally infected with IBDV not only could become the carriers or reservoirs in transmis—sion but may take certain effect on the variation of IBDV.

cDNA Library Construction of a Chinese mitten crab reovirus RNA1 and Partial Sequence Analysis of its RNA Polymerase Gene

ZHANG Shu—yonga.Bonami Jean-Robert, SHI Zheng-li ”

2003, 18(1): 72

A reovirus (designated as EsRV905), was isolated from Chinese Mitten Crab(Eriocheir sinensis). Viral genome was extxacted with Trizol reagent and individual segment was recovered separately from gel after 6.5% PAGE. The cDNA library of RNA1 was synthesized using random primer method and short fragments l ess than 200 bp were discarded using gel recovery kit. Th e cDNA was ligated to blunt-end plasmid and transformed to competent cel1. Positive clones were screened by blue and white colonies and checked by enzyme digestion. Two plasmids containing around 1.5kb inserts from the first segment were selected for sequencing. One of them contained the main motif of RNA polymerase(RdRp).The RdRp of EsRV905 is located in the first segment of EsRV905 genome.

Detection of Wheat yellow m osaic virus by Heter0gene0us Animal Double—antibody Sandwich ELIS:A

GENG Bo, HAN Cheng-gui”, ZHAI Ya-feng, WANG Hui-qing, YU Jia—lin, LIU Yi

2003, 18(1): 76

Abstract:With purified Wheat yellow mosaic vires(WYMV) particles as antigen, cavy was injected to produce specific antiserum.Its titer was tested as Y1 28 by the method of micro-immunoprecipitation. Using cavy antiserum as capture antibody for trapping the virus and rabbit antiserum as detection antibody, the experiment system of heterogeneous animal DAS-ELISA (HADAS—ELISA)was established to detect WYMV.The system has the advantage of better specificity and sensitivity of detection.This method was proved to be successful and prattical in the detection of the virus in field—-collected sam-· ples, indicating that the experiment system of HADAS—ELTSA is superior to the indirect ELISA with rabbit antiserum only.

Genetic Analysis of Simian virus 40 from Peripheral Blood and Kidney of M acaque M onkeys

LIU Jian—sheng, LIU-Xin, XU Dong—lei, SHAO Cong—wen, HOU Zong—li

2003, 18(1): 79

In order to investigate the detection methods and gene structure of SV40 ST-ag and LT—ag— C in peripheral blood mononuclear cells (PBMC) and kidney cells from different Macaque monkeys, using PCR, 4 positive templates were obtained firstly, then 4 ST—ag gene fragments and 4 LT—ag—C gene fragments were achieved and sequenced. These antigens coding region of each isolate contain a number of nucleotide differences when compared to the reference strain SV40——776 and the laboratory strain SV40一B.These resuhs indicated that:1)SV40 DNA isolated from the PBMC and kidney cells showed essentially identical sequences for ST—ag gene and LT—ag—C gene. 21 the presence of viral DNA in P—BMC suggests that virus may spread with in the host by hematogenous routes. 3) a new SV40 strain predominates in our monkey population, it contains a specific nucleotide and amino acid changes in LT-ag—C termini, TCCTCACAG polynucleotide is inserted among nt2784/2783, SO SerSerGIn iS inserted after amino acid 678 residue.

Advances in Aquareovirus Research

FANG Qin, ZHU Zuo-yan

2003, 18(1): 82

Recent Advances on M olecular Biology of Potato virus X and Its Application to Gene Expression Vector

QU Jing, GUO Xing-qi, CI Xiao-yan, QI Dong, WEN Fu-jiang*

2003, 18(1): 87

Advance in Avian leukosis virus Subgroup J

YANG Yu-ying

2003, 18(1): 93