MO Xue-mei, YE Shi-dun, ZHANG Guang and SUN Han-xiao*. Anti-SIV Potency of a Broad Spectrum Chemokine Receptors Inhibitor，vMIP-II In Vivo[J]. Virologica Sinica, 2005, 20(5): 459-463.
Viral macrophage inflammatory protein II(vMIP-II) encoded by Human herpesvirus 8 is a potent antagonist of various chemokine receptors,which are believed to be co-receptors for Human immunodeficiency virus(HIV) entry of different strains.Although it is known as a broad-spectrum HIV entry inhibitor,the exact anti-HIV mechansim of vMIP-II has been rarely reported,especially in vivo studies.In the present study,the well-established SIVmac251-infected cynomolgus monkey model was used to study the anti-HIV potential of vMIP-II in vivo.It was shown that vMIP-II caused rapid and significant decrease in plasma viral loads in a dose-dependent manner,which was comparable to that of positive control.Meanwhile,vMIP-II was found to protect the host immune function.In summary,these results indicate that vMIP-II is a potent anti-HIV agent and provides further rational to develop entry inhibitor.
CAI Heng-ling, WU Yi-mou, WAN Yan-ping, XIAO Jian-hua, YANG Qiu-lin, ZENG Qiao, ZHAO Fei-jun and TANG Man-juan. Construction of the Eukariotic Expression Vector for s1 Gene Fragment of SARS-CoV and its Immunol ogical Effects[J]. Virologica Sinica, 2005, 20(5): 464-467.
The 801 base pairs of S1 gene fragment were synthesized based on the Sever acute respiratory syndrome associated coronavirus(SARS-CoV)BJ01 strain registered in GenBank.The synthetical DNA was subcloned into the appropriate site of pcDNA3.1(+) eukariotic expression vector to construct a recombinant plasmid pcDNA3.1(+)/S1;The recombinant plasmid pcDNA3.1(+)/S1 was transfected into Hela cells and the expressed protein was identified by SDS-PAGE and Western-blotting;BALB/c mice were immunized with pcDNA3.1(+)/S1 by i.m.The level of anti-SARS-CoV IgG and IFN-γ in BALB/c mice after immunization were detected by ELISA and T cell proliferation activity was tested by MTT.It was found that the recombinant plasmid pcDNA3.1(+)/S1 could express S1 protein in Hela cells,T cell proliferative activity,the level of anti-SARS-CoV IgG and IFN-γ increased after immunization.It revealed that pcDNA3.1(+)/S1 could induce moderate cellular and humoral immunological reaction in BALB/c mice.
MENG Xian-feng, DAI Xing, MENG Ji-hong, ZHANG Hong-mei, ZHOU Zhen-xian, Genotype Identification and Variation Analysis of Hepatitis E Virus Isolated in Nanjing, China[J]. Virologica Sinica, 2005, 20(5): 468-471.
The objective of this study was to identify and analyze genotype distribution and genetic variation of Hepatitis E virus(HEV) isolated in Nanjing,China.RT-nPCR based on universal primers for multiple HEV genotypes was used to detect HEV RNA in 40 samples collected from patients with acute sporadic hepatitis E.PCR products were sequenced and analyzed with the LASERGENE and PHYLIP softwares.Fourteen out of the 40 samples were positive for RT-nPCR.The positive rate was 35%.All of the fourteen isolates were clustered into HEV genotype IV and were separated into two different sub-genotypes.The nucleotide sequences of the HEV isolates of genotype IV presented higher variability when compared with the sequences of the Chinese isolates of HEV genotype I.Moreover,a new subtype of HEV genotype IV was discovered in this study.
MA Cui-qin, ZHOU Yu-sen, WANG Yi-jia, GUO Yan, GUAN Jie, WEI Lin and HE Yu-xian. Expression and Immunogenicity Analysis of the Receptor-binding Region of SARS-CoV Spike Protein[J]. Virologica Sinica, 2005, 20(5): 472-475.
In order to express the recombinant protein of SARS-CoV S receptor-binding region and to study its immunogenicity,the gene encoding SARS-CoV S receptor-binding region was cloned to pET32a+ and expressed in E.coli.The expression of the recombinant protein was confirmed by Western blot analysis.The S purified protein as diagnostic antigen coated the microtiter plates to detect 20 SARS sera and 28 healthy sera.The results showed that the S purified recombinant protein was able to react with all sera of SARS patients.BALB/c mice were immunized with the denatured and refolding protein respectively.Sera were collected after the third immunization for detection of neutralizing activity against SARS virus.The denatured recombinant S receptor-binding region protein could not induce neutralizing antibodies,whereas refolding recombinant S-RBD could elicit neutralizing antibodies.These results indicated that the receptor-binding region of the S protein could be used for vaccine development.
DENG Chun-qing, DENG Guo-hong and WANG Yu-ming*. eNOS Gene (894G/T) Polymorphisms Among Patients Infected With HBV[J]. Virologica Sinica, 2005, 20(5): 476-479.
To characterize the relationship between exon7 of eNOS gene(position 894G/T) polymorphism and clinical subgroups infected with HBV.The genotype was analyzed by using method of polymerase chain reaction(PCR),enzyme restriction after PCR amplification,and agrose gel electrophresis.Results show that statistic differences of genotype and allele frequency exist in the patients between AsC(asymptomatic carrier)and any other subgroups;between AsC+CHB(Chronic hepatitis B)and SHB(Severe hepatitis B)+LC(Liver cirrosis) subgroups.Logistic regression analysis supports that G/G genotype of exon7 is more succeptiable to develop Asc state than G/T or T/T genotype.So exon7 of eNOS gene(position 894G/T) polymophism exists among the people infected with HBV,espescially G/G genotype of exon7 is might more succeptiable to develop Asc state than G/T or T/T genotype.
MA Jia, WANG Rui-qing, SONG Jian-hua, LIANG Chang-yong and CHEN Xin. Expression and Localization of the HCV Structural Protein E1 in Insect Cells[J]. Virologica Sinica, 2005, 20(5): 480-484.
In this work,the truncated Hepatitis C virus(HCV) structural protein E1 fused with GST was expressed in E.coli.The fusion protein was used as an antigen to immunize rabbit to produce the antibody for detection.To express HCV E1 in insect cells,the recombinant baculovirus(vAcHCVE1) containing the HCV full length E1 gene was constructed by Bac-to-Bac recombinant baculovirus expression system.It was proved to be able to produce HCV structural protein E1 in insect cell Sf9.The expression protein was detected by Western blot analysis.The size of the product was 30 kDa,which was bigger than the predicted size of 20 kDa,implicating post-translational modification such as glycosylation.By using confocal microscope,it was observed that the E1 protein was localized in the cytoplasm and cellular membrane 48h after inflection.
HUANG Wei-jin, WANG You-chun* and ZHANG Hua-yuan. Expression of Fusion Gene Containing HBV S, pre-s1 and C Fragments in Yeast and Analysis of its Anti- genicity[J]. Virologica Sinica, 2005, 20(5): 485-489.
PCR method was used to Construct multi-epitope gene ss1c containing HBV S(1-222AA),preS1(10-50AA)and C(1-30AA) gene.The fusion ss1c gene was cloned into the expression vector pPIC3.5k.The recombinant vector was transformed into the host cell of GS115 with electroporation method.After screening with G418 and inducing with methanol,the product was analyzed with Western blot and EIA.The fusion gene(SSIC) with the length of 906bp was successfully expressed in yeast system.The molecular weight of SS1C expressed protein was about 31kDa and virus-like particles with diameter of about 22nm were formed.The product had specific reaction with anti-HBs,anti-preS1 and anti-HBc mAbs.Whether SS1C is a promising candidate for therapeutic vaccine for control of chronic HBV infection needs to be further studied.
PENG Yu, LI Shu, LI Chao-yang, WANG Jing-jing, TIAN Bo and GUO De-yin*. Interaction between Nucleocapsid Protein and the Leader the Sequence of SARS-CoV RNA[J]. Virologica Sinica, 2005, 20(5): 490-493.
SARS coronavirus(SARS-CoV) is a novel human coronavirus and was responsible for SARS outbreak in several regions of the world in 2003.The genome of SARS-CoV is about 30,000 nucleotides in length and sequence analysis revealed that nucleotides 1-72 contain a RNA leader sequence preceding an untranslated region(UTR).In order to confirm the interaction between the N protein and the RNA leader sequence,the N gene was cloned,sequenced and the protein was expressed in Escherichia coli as a fusion protein with His-tag.The recombination N protein was purified by Ni-NTA affinity column and G-75 column.Meanwhile,the DIG labeled RNA leader sequences were synthesized by in vitro transcription.RNA-Protein interaction was detected by Northwestern RNA blot assay.The results indicated that a specific interaction takes place between the N protein and RNA leader sequence.Our results provide some insights into the understanding of assembly of the SARS-CoV particles,which is an essential step for viral replication cycle.
ZHANG Xue-han, HE Kong-wang, GUO Rong-li, NI Yan-fang, WANG Fang and YU Zheng-yu. Cloning and Expression of E Gene of Japanese Encephalitis Virus and Preliminary application[J]. Virologica Sinica, 2005, 20(5): 494-497.
The whole cDNA of E gene was amplified by RT-PCR from JEV strain SA14-14,and cloned into the pMD18-T vector.The fragment was identified by restriction enzymes digestion with EcoR I and Xho I,cloned into the pET32a(+) vector.The recombinant plasmid was transformed into BL21 and the recombinant bacteria was induced by optimal concentration of IPTG.SDS-PAGE and Western blotting were performed to detect the E fusion protein.Our result indicates the molecular weight of E protein is of E protein 73kDa and its antigenicity is good.Indirect ELISA procedure has been developed with the purified E protein as antigen for detection of JEV.
HUANG Bing, MA Xiu-li, WANG Li-li, LI Yu-feng, LIU Yu-shan and CHEN Fu-uan. Rapid Identification of Hemagglutinin Gene of Avian Influenza Virus Subtype H9 and H5[J]. Virologica Sinica, 2005, 20(5): 498-502.
A method of one-step reverse transcription polymerase chain reaction was developed to subtype hemagglutinin(HA) gene of Avian influenza virus(AIV).One pair of primers were designed on the basis of HA gene of AIV,allowing simultaneous detection of AIV subtype H_9 and H_5,and the specific amplicons were 579bp and 177bp in size respectively.The primers were specific for AIV(H_9 and H_5 subtype) with no cross-reaction to RNA from the chicken muscle and other chicken pathogens such as Newcastle disease virus etc.The sensitivity of this assay was about 50pg of total RNA of AIV.Our data showed that this method was simple and feasible for rapid identification and subtyping of AIV subtype H_9 and H_5 at a single reaction.In addition,both diagnostic amplicons for AIV subtype H_9 and H_5 contained the cleavage site sequence of HA gene,from which the amino acid sequence might be deduced to predict the potential virulence of AIV.
WANG Yong-shan, FAN Hong-jie, LI Yin, ZHOU Zong-an, SHI Zheng-liang, WANG Xuan-nian and ZHANG Gai-ping. Identification and Sequencing of Five Peptides Containing Epitopes of Infectious Bursal Disease Virus[J]. Virologica Sinica, 2005, 20(5): 503-506.
Five monoclonal antibodies to Infectious bursal disease virus(IBDV),HNF1,HNF7,B34,2B1 and 2G8 were used to screen for binding peptides from peptide 12-mer phage display library.After three rounds of panning(absorption-elution-amplification),sixty positive monoclonal phages(twelve for each monoclonal antibody) were selected and the phage displayed 12-peptides were detected and identified with indirect ELISA(A value 1.00) and competitive inhibition ELISA(inhibition rate 40%).The results indicated that 12-peptides contained epitopes of IBDV.Thirty-five positive monoclonal phages were sequenced,and the sequences of nucleotides and amino acids of the five different epitopes on IBDV were determined and analyzed.Comparison with sequences of IBDV registered in GenBank,four continuous amino acid residues Leu-Ala-Ser-Pro of 2B1 selected peptide was homology with the sequence encoded by genome fragment A from 536 to 539.But HNF1,HNF7,B34 and 2G8 selected peptides had no more than three continuous amino acid residues s
LI Pei, CAO Rui-bing, ZHOU Bin, ZHENG Qi-sheng, WEI Jian-chao, LI Peng, REN Xue-feng and CHEN Fu-yan. Prokaryotic Expression of the GP3 Protein of Porcine Reproductive and Respiratory Syndrome Virus S1 Strain[J]. Virologica Sinica, 2005, 20(5): 507-510.
The recombinant plasmid pET-VP_2Ⅰ was transformed into BL21 competent cells and expressed in high level after induced with 1.0mmol/l IPTG.The expressed product was purified with His·Bind chromatograghy after being proved by Westen-blot,and was used as an antigen to establish indirect ELISA for detecting antibodies against PPV.The optional working circumstances for the ELISA(antigen concentration 3.5μg/mL;serum dilution 1∶40)were tried out with chess titration.The positive critertion of test serum of this ELISA is OD_490nm0.51,the OD_490 ratio of the tested serum to the negative serum is higher than 2.1.
JANG Yi-jun, JIANG Ping*, YANG Xiao-wei, TANG Jing-yuan, LI Yu-feng and LI Yong-dong. Construction and Indentification of Recombinant Adenovirus Containing Multiple Antigen Epitopes of Swine Foot-and-Mouth Disease Virus[J]. Virologica Sinica, 2005, 20(5): 511-514.
A recombinant replication defective Human adenovius serotype5 plasmid pAd2VP ,containing amino acids (21260)2(1412160)2(2002213) coding region of swine foot and mouth disease virus serotype O-VP1 , was const ructed using the method of homologous recombination in E.coli BJ5183. After the recombinant plasmid pAd2VP linearized with PacI t ransferred into HEK293A cell , the recombinant virus was isolated and purified in HEK2293A cells by t hree times plaque purification . This recombinant adenovirus could be stably passaged in HEK2293A cells and TCID50 was 10
SONG Jian-ling, WANG Jin-ping, HU Yuan-yuan and ZHANG Fu-qiang*. Expression of M1 protein of Avian Influenza Virus and Analysis of Its Immunoreactivity[J]. Virologica Sinica, 2005, 20(5): 515-518.
A pair of primers were designed based on M1 gene sequence of known H5N1 Avian influenza virus.M1 gene was cloned from total RNA,extracted from tissue of H5N1 subtype virus inoculated embryo by reverse transcriptase-polymerase chain reaction using high proofreading polymerase(Pyobest~TM DNA Polymerase),and expressed using Invitrogen champion~TM pET directional TOPO expression system.Recombinant protein containing polyhistidine(6xHis) tag in N-terminal about 29.8kDa in size,wac obtained and purified.Its immunoreactivity was analyzed by Western blot,ELISA and blocking ELISA using MAb and positive serum.The results showed recombinant M1 protein can bind to MAb and positive serum with specificity,and this binding reactivity can be blocked by natural viral antigen.This indicates that the recombinant M1 protein possesses good immunoreactivity.
JIANG Wen-ming, JIANG Ping* and LI Yu-feng. Prokaryotic Expression of the GP3 Protein of Porcine Reproductive and Respiratory Syndrome Virus S1 Strain[J]. Virologica Sinica, 2005, 20(5): 519-521.
The GP3 deleted hydrophobic N-terminal sequence(tGP3) of Porcine reproductive and respiratory syndrome virus(PRRSV) was cloned into prokaryotic expression vector pRSET.The recombinant protein(His)_6-GP3 was highly expressed in E.coli BL21 and could amount to 41% of the total proteins.It could be purified efficiently with affinity chromatography.Western-blotting analysis showed that the recombinant protein was able to react with PRRSV polyclonal antiserum.It can be used for the further investigation on immunogenicity and function of GP3 of PRRSV.
WU Jian-min, YANG Yu-biao, LV Mao-min, GUO Yan-ru, ZHANG Jin-gang and Cloning of Porcine Endogenous Retrovirus 5￠-Untranslated Region and Analysis of Its Structure[J]. Virologica Sinica, 2005, 20(5): 522-525.
The full-length 5′-untranslated region(5′UTR) of Porcine endogenous retrovirus(PERV) from Chinese Wuzhi shan(WZS) miniature pigs was cloned by RACE.Seguencing of PERV-WZS 5′UTR showed that it was 1035bp.Compared with other PERV 5’UTR sequences published in GenBank,the homology was 82.6~94.8%.Primary structure analysis revealed that the PERV-WZS 5′UTR was composed of U3,R,U5,primer binding site(PBS) and leader sequence.The potential promoter and enhancer of the 39-bp repeat sequences were located in U3 region(-67~+1) and(-97~-59) respectively.There were 31 potential cis-acting elements such as NF-Y,TBP,Oct-1,HSF,GATA-1,and GATA-2,which were considered to be related to PERV transcription and regulation.
WANG Bao-qin, WANG Xiao-long, ZHANG Yong-guang, PAN Li, WANG Wen-xiu and DONG Jin-jie. Transformation of vp1 Gene of Foot-and-Mouth Disease Virus in Legume Forage Lotus cornicu- latus L[J]. Virologica Sinica, 2005, 20(5): 526-529.
The cotyledons and cotyledon petioles of legume forage Lotus corniculatus L.were transformed by Agrobacterium tumefaciens-mediated method with the binary expression vector pBinFMDV-VP1,which contained the vp1gene of FMDV strain Akesu/58 serotype O.Kanamycin-resistant plants were obtained after selected with 50 mg/L kanamycin.The vp1gene was analyzed through PCR with total DNA and through RT-PCR with total RNA from fresh leaves tissue of the kanamycin-resistant lines,the random samples from positive Lotus corniculatus L.lines were analyzed by Western-blotting.These results showed that the vp1 gene was transformed into Lotus corniculatus L.plants and had transcription activity;the VP1 protein was expressed correctly.The transgenic lines was transplanted to greenhouse,and further work is under way.
HUANG Juan, JIANG Ping, ZHANG Chang-yin, TANG Tai-shan, LI Yong-dong and ZHANG Zhi-tao. Development of Real-time PCR for Detection of Porcine Reproductive and Respiratory Syndrome Virus[J]. Virologica Sinica, 2005, 20(5): 530-533.
Primers and probe were designed from the nucleotide sequence within the ORF7 of Porcine reproductive and respiratory syndrome virus(PRRSV),and a quantitative TaqMan real-time PCR was developed for PRRSV detection.Specificity and sensitivity were compared with virus isolation and a conventional RT-PCR,and were found to be equal or superior to the reference methods.Reproducibility was tested and proved that the assay was reliable.Standard dilutions allowed absolute quantitation of the amount of viral RNA.The TaqMan real-time PCR described below is time-saving,easy to handle,exhibits a decreased risk of cross-contamination and is highly sensitive and specific.It is considered to be a powerful tool for the rapid detection of PRRSV.
ZHANG Hai-yuan, HUANG Bo-qing, MEI Chun-lei and ZHANG Zhong-xin. Cloning，Expression and Function Analysis of Cathepsin Gene Encoded by Spodoptera exigua Nucleopolyhedrovirus Cloning，Expression and Function Analysis of Cathepsin Gene Encoded by Spodoptera exigua Nucleopolyhedrovirus[J]. Virologica Sinica, 2005, 20(5): 534-538.
A putative v-cathepsin(v-cath) gene of Spodoptera exigua nucleopolyhedrovirus(SeMNPV) was cloned,sequenced and expressed.The results showed that the gene size was 1014 nucleotides,encoding a 338 amino acids putative protein.The amino acid sequence alignment of ten baculoviral v-CATHs showed that the primary structure and the catalytic sites were conserved.A pholygenetic tree of these v-CATHs constructed by using maximum parsimony analysis indicated that the v-cath gene of SeMNPV had a different evolutionary history from that of other NPVs.When late 3th instar Spodoptera exigua larvae were infected with SeMNPV combinded with 1.2 μL expressed bacteria and medium,the mortality was increased 9.21%,and LT50 was 12 h shorter than the control.The v-CATH expressed in prokaryote has a significant effect on the killing speed of SeMNPV insecticide.
FAN Guo-cheng, WU Zu-jian, HUANG Ming-hua, LIAN Jun, DONG Liang, LIN Qi-ying and XIE Lian-hui. Molecular characterization of the genome segment S9 of Rice gall dwarf virus[J]. Virologica Sinica, 2005, 20(5): 539-542.
The full-length cDNA of the genome segment(S9) of Rice gall dwarf virus Xinyi isolate(RGDV-C) was cloned and the complete nucleotide sequence was determined.The results showed that S9 had 1202 nucleotides(AY556483) encoding a polypeptide of 323 amino acids with an Mr of 35.6kDa.The S9 had the same genomic organization as that of RGDV Thailand isolate(RGDV-T) and shared 98.1% nucleotide and 98.5% amino acid sequence identity.No equivalent protein of Pns9 encoded by RGDV S9 ORF was found in other members of the genus Phythoreovirus.Pns9 shared 21.8% amino acid sequence identity with ATP-dependent Clp protease proteolytic component from Borrelia burgdorferi.
CHE Hai-yan, LUO Da-quan*, YANG Xu-guang, FU Rui-yi and YE Sha-bing. Detection and Partial Sequence Analysis of Whitefly-transmitted Geminivirus in Carica papaya and Hibiscus rosa-sinensis in Hainan[J]. Virologica Sinica, 2005, 20(5): 543-545.
Three virus-infected Carica papaya samples showing mosaic,leaf roll symptoms and three virus-infected Hibiscus rosa-sinensis samples showing chlorosis and distortion symptoms were collected in Danzhou of Hainan Province.A pair of degenerate primers was designed according to the conserved sequence of CP gene and intergenic spacer of Whitefly-transmittal geminiviruses(WTGV),PCR product of expected size was amplified from six samples.The specific fragments were cloned and sequenced,and sequence analysis showed that PCR product had a geminiviral gene origin,which was demonstrated by BLAST.Partial DNA-A between P1 and H2 shared only 66% nucleotide sequence identity.Polygenie analysis with DNA-A of P1 and H2 from other 16 WTG,P1 was most closely related to LAYVV,H2 differed from those of other geminiviruses and constitute a new evolutionary branch.
HUANG Xin-xin, MO Sheng-lan and LU Cheng-ping*. RT-PCR Detection of Taura Syndrome Virus in Penaeus vannami from Southeast Seaside of China Virus[J]. Virologica Sinica, 2005, 20(5): 546-548.
Taura syndrome virus(TSV) is one of the important pathogenic agents of Penaeus vannami,which causes prawn infection with serious disease in China in recent years.245 samples collected from 26 farms of Zhangzhou,Xiamen,Shenzhen,Yangjiang,Ningbo and Guangzhou.were detected for TSV with RT-PCR,the positive rates were 100%,33.3%,40.7%,50% and 40%,respectively.Southern blotting and nucleotide sequencing were performed to confirm the specificity of the amplified RT-PCR products.The homology of 231bp nucleotide sequences reached 98.3%-100% when analysed and compared with the GenBank using the BLAST program.Phylogenetic analyses clustered the TSV isolates into two groups: one contained USA and Chinese Taiwan isolates;the other contained Vietnam isolate(YN1) and 21 isolates of mainland China.The 21 China isolates clustered into one branch,while the YN1 distributed in another branch.5 strains from Guangzhou and 2 strains from Shenzhen constituted one subcluster of the China branch.
XU Xin-gang, HU Jian-he, ZHANG Yan-ming and DENG Hong-kui. Construction of Hepatitis C Virus E1E2 Envelope Protein Gene DNA Vaccine and Animal Immune Experiment[J]. Virologica Sinica, 2005, 20(5): 549-551.
Hepatitis C virus(HCV) H77 strain E1E2 gene DNA vaccine was constructed by inserting full-length cDNA of HCV E1E2 into an eukaryotic expression vector pcDNA4.0.The recombinant plasmid was transfected into eukaryotic cells 293T by calcium phosphate transfection method and transient expressed E1E2 envelope protein was analyzed by FACS.BALB/c mice were injected intramuscularly with the recombinant plasmid.Anti-HCV E1E2 antibody was detected by FACS with SP2/0 cells which expressed HCV E1E2 protein.Moreover,the antibody was also analyzed by Westrn blot using prolyofic expression E2 protien as the arigen.The results showed that HCV E1E2 protein was expressed transiently in 293T cells.Specific anti-E1E2 antibody could be detected in DNA immune mouse serum by FACS and the antibody could react specifically to SP2/0 cells which express HCV E1E2 protein.Western blot analysis showed that DNA immune mouse serum could react specially to E2 protein expressed in E.coli.
LIU Jia-sen, CHEN Shu-hong, LIU Huai-ran, PANG Hai, KONG Xian-gang and Prokaryotic Expression of SARS Coronavirus Nonstructural Protein 9[J]. Virologica Sinica, 2005, 20(5): 552-554.
The gene of nonstructural protein 9 of SARS-coronavirus(SARS-CoV) was amplified using PCR from the product derivated from reverse transcription of SARS-CoV genome RNA,and was inserted into the multiple cloning sites of the expression vector pGEX-6p-1.Recombinant strain induced with IPTG expressed the specific soluble protein.The nsp9 protein was harvested and purified by affinity chromatography and processed by prescission protease.Polyclonal serum against the nsp9 protein was raised in rabbit.
ZHANG Ping-hu, XUE Feng, TANG Ying-hua, QIAN Zhong-ming, HAO Gui-jie, LIU Hui-mou and LIU Fan-xiu*. Molecular Analysis of the Surface Glycoprotein Genes of an Aquatic Bird Influenza Virus[J]. Virologica Sinica, 2005, 20(5): 555-557.
Several H11N2 subtype of Avian influenza A viruses were isolated from aquatic birds in live bird markets when we surveyed the ecology of the influenza in East China for more than two years and identified by specific RT-PCR.The hemagglutinin(HA) and neuraminidase(NA) genes of one representative virus named A/Duck/Yangzhou/44/2002(H11N2)(DYZ/44/02) was sequenced.The results showed the HA nucleotide sequence of DYZ/44/02 has high identity with Dk/England/56(H11N6) and the NA nucleotide sequence of DYZ/44/02 has more than 95% sequence homology with Dk/Hokkaido/49/98(H9N2).Sequencing and phylogenetic analysis of the N2 NA genes of DYZ/44/02 revealed that the NA gene of DYZ/44/02 has close relationships with that of Ck/Korea/MS96/96-like H9N2 virus and are distinct from those of Ck/Beijing/94(H9N2).The sequence of cleavage site of DYZ/44/02 consists of a single arginine,as is the case with most other hemagglutinins exhibiting low susceptibility to proteolytic activation.