2003 Vol.18(3)
Abstract： Wle examined 121 Specimens of exfoliated cells and tissue of cervix for the presence of肿V一16／18 an d HSV一2 DNA by PCR．At the same time，we analysised 76 specimens from tissue ofcervix for the expression of p53 by immunohistochemical assay．The HPV一16／18 was found in 19(61．3％)of 31 cervical carcinoma，in 9(22．5％)of 40 chronic cervicitis，in 3(6．O％)of 50 normaluterine cervix．The positive rate of HSV一2 was 32．3％ (10／31)，2O．O％ (8，4O)and 4．0％ (2／50)．1 emixed infection rate of肿V 16／18 an d HSV一2 was 16．1％ in cervical carcinoma．The rates of positiveexpression ofp53 was found to have a gradient of crescendo in the tissues of chronic cervicitis．CIN an dcervical carcinoma．Wle found that cerv ical carcinoma is strongly related witll HPV一16／18 infection．Asynergetic action may OCCUr between HPv an d HSV一2 infections in the oncogenesis of cervicalcarcinoma．Th e overepression of p53 appears to be closely associated with the development of cervicalcan cer．Th ere is a matter ofclinical significan ce on the prevention an d treatment ofcervical carcinoma．
The gene encoding a strategically truncated E glycoprotein，approximately 80％ of the N·terminal sequence was cloned into the eukaryotic expressing plasmid pCXN2 to get the recombinant plasmid pCXN2一E．Indirect immune fluorescence assay showed that the recombinant plas mid pCXN2·E Can be expressed in COS一7 cells．Th e specific an tibody against DV2 E was detected by ELISA at 2 weeks after the last inoculation，an d maintained to 1 5 weeks；Plague red uction neutralization test (PRNT)was performed on sera obtained at 2 weeks post inoculation．Th e sera had high PRNT50 titer which was overl"640；Th e percentage of CD4+T—lymphocyte subtype cells in immune mice increased compared with the control group．Th e percentage of CD8 T—lymphocyte subtype cells in the group of pCXN2一E was significantly higher than that of pCXN2 group (p0．01)；Mice protection against challenge showed that 60％ mi ce survived．In conclusion：pCXN2一E Can induce humoral an d celIuar immune response．Especially,the raise of CD8 CTL is importan t to clear virus．
The E2 gene of Hepatitis C Virus was amplified by PCR．and cloned into the pQE30 at the downstream of LacZ promoter．The recombinan t vector DNA was introduced into the E．coli盯 109 cell to create the JM109 pQE2．The HCV E2 protein fused to 6 His peptides with molecular weight of 35kDa was produced in JM109 pQE2 cel1．To study the immunological property of E2 protein．the rabbit and BALB／c mice were vaccinated with E2 protein purified by Ni．NTA．Superflow．The antibody against E2 was detected by ELlSA，the results showed that the HCV E2 specific an tibodies could be observed at the 14th day after immune．then the level of an tibodies rose an d reached the highest pe ak at the 55m day．then the an tibodies had maintained at this high level with titer of 1：3200 in this study．The specific CTLs were gotten from the mice spleens，after simulated an d augment in vitro，they were used as the effect cells to ki11 the target cells，which were called P8 15 tran sfected wtih pCE2．By LDH assay， we can find that more than 30％ of the target cells were killed at the ration E：T=20o：1．an d there are diflferent effects in diflferent immunity injection means．Th e results show that the E2 protein Can provoke an tibody in immune rabbits an d specific CTLS in the mice．From this study．we suggest that the E2 protein is a good can didate for developing a vaccine to prevent HCV infection
Abstract：1]he neuraminidase(NA)gene of Influenza virus A／FM，1／47 was amplified by RT—PCR and cloned into adenovirus vector pTrack—CM V The recombinan t plasmid an d adenovirus DNA Was cotransformated into E coli BJ5 1 83 an d the recombinan t adenovirus genomic DNA Was obtained through homological recombination．The recombinan t adenovirus was gained after tran sfecting 293 cell line with the genomic DNA．The intergration of NA in resultan t adenovirus an d the expression of NA gene were confilrned by PCR an d western—blot respectively．BALB／c mice were immunized intranasaUy and orally respectively．ELISA result showed that systemi c immue responses to Influenza virus could be induced efectively in both intran asal an d oral route．Th e immunization efficacy of intran asal route Was superior to that of oral route．
To investigate the function of human rotavirus nonstructrual protein NSP4 in the pathogenesis of rotavirus, the cDNA of NSP4 gene of a human rotavirus strain 97SZ8 was cloned and its 86-175 amino acid at the C terminal fused with GST was expressed in E.coli BL-21, the recombinant protein was then purified with Glutathione Sepharose~(TM) 4B affinity chromatography. Purified NSP4 protein was administered to young (6d) inbred Balb/C mice by the intraperitoneal (IP) route (0.4nmol or 1.5nmol). IP administration of 0.4nmol recombinant protein induced diarrhea in just 1 of the 6 pups, while diarrhea was observed in all of the 12 pups when inoculated with 1.5nmol GST-NSP4_T and resulted in reduced weight gain (P0.05). In this study, a mouse model of diarrhea induced by human rotavirus NSP4 was successfully developed, and can be used widely in the highlights on mechanisms of diarrhea, treatment and prevention of human rotaviruses.
Abstract：XJ一1 60 is the first Chinese isolate of a Sindbis—like virus，an d its genome was completely sequenced in our previous work．In this investigation，a full—length genomic cDNA clone Was firstly constructed from RT—PCR products of the viral RNA．Then．the plasmid of replicon expression vector (pRepxj 1 60)was derived from this cDNA clone replacin~viral structural sequence with multiclonal seq uence by DNA recombination technique．To verify function of this vector,reporter genes．EGFP an d cZ．were cloned into this plasmid．respectively．And RNA tran scripted from expression plasmid Was inducted into BHK一2 l cell line． resulting in expression of green fluorescent protein an d 13 ．galactosidase 14 hours later．Th e results indicated mat the replicon vector derived from )(J—l60 virus was self-replicating and that the following gene expression was efficient．Our study settled the basis for developing all：Ihavirus vector system with Chinese intellectual property．
Mechanism of oncolysis of Newcastle disease virus (NDV) CN strain is studied in vitro in five carcinoma cells including HEP3B, T24, A549 and Hela cells, The pseudopods of the infected cells were retracted after 16h infected with 16HU/mL CN strain, the cells changed round and lost adhesion, the survival rates of the infected cells were lower than 10% by 120h. HEP3B cells was the most subjective to NDV. But CN strain seldom killed human normal cells 2BS. At early stage of infection (16h after infection) virus nucleic acid coding HN was detectable in cytoplasma by RT-PCR, NDV antigen was detectable by immunofluorescent test. At the same time, inspection of electric microscope showed NDV CN strain induced apoptosis in HEP3B and T24 cells. And induced necrosis in Hela, Hep2 and A549 cells.
According to PCR primers for SARS developed by WHO network laboratories we synthesize 4 primer pairs: BNIin-s/as、BNIout-s/as、SARl-s/as、COR1/COR2 and establish assays of one-step RT-PCR for detection of SARS associated coronavirus. Specific DNA fragments as predicted were produced from specimen W20 by the assays using all 4 primer pairs, while one specific DNA fragment was produced from specimen W1 using primer pair BNIin-s/as. The PCR products from W20 were sequenced and the sequence showed high identity to that of all SARS associated coronavirus in GenBank, which showed that PCR products amplified from W1 and W20 by our assays was specific for SARS associated coronavirus. Based on the one-step RT-PCR we developed four one-step duplex RT-PCR for amplifying two fragment in one reaction by combination of 4 primer pairs above. And the assays of one-step duplex RT-PCR have higher specificity and were reliable for detection of SARS associated coronavirus.
Sublethal effects of reproduotion in female adults were found when eighth and ninth-instar female P. fuliginosa nymphs werw treated by PfDNV with three diffirent concentrations. The results showed that the longevity of female adults were reduced 121days, 145days and 179days, respectively. The cviposition period were reduced 62days, 56days and 121days, respectively. The number of oothecaes laid were reduced 16 oothecaes/female, 20 oothecaes/female and 25 oothecaes/female, respectively. The number of eggs laid were reduced 354 eggs/female, 453eggs/female and 558 eggs/female, respectively. The oothecae viability dropped 28%, 28% and 20%,respectively. The fertility dropped 177 times, 226 times and 279 times, respectively. The female adults of P. fuliginosa were effected by DNV when vigorous period of eggs laid in early 4 months continuously. Mean number of egg laid per month were reduced 30eggs/female, 69 eggs/female, 63eggs/female and 55 eggs/female,respectively. Fertility per month dropped 15 times, 34 times,31
Wild HaNPV-VHA_(273) Multi-nucleocapsid NPV and its clone Single-nucleiocapsid NPV H_9 were compared on the shape and structure, biological activity, restriction pattern and structural polypeptide.The LC_(50) values of the wild and clone isolates for early third instar H.armigera larvae were 2.987×10~4 PIBs/mL and 1.647×10~4 PIBs/mL respectively; The LT_(50) values for 2×10~7 PIBs/mL concentration were 4.866 and 4.797 days respectively. Bioactivities of two isolates were similar. Assayed by SDS-PAGE, there were more difference between VHA273 and H_9 structural polypeptides.Digested by EcoRI, Bam HI, Hind Ⅲ and Xba Ⅰ, VHA_(273) and H_9 genomes had some differences in their fragmentation profiles. This discovery will be helpful for revealing the factor what caused the Multi-nucleocapsid NPV and Single-nucleocapsid NPV’s formed in molecular level.
Abstract：Dendrolimus punctatus cytoplasmic polyhedrosis virus(DpCPV)genome dsRNA wag extracted an d S4 was separated．RT—PCR amplification technique Wag adopted to clone the viral genomic dsRNA S4 flor sequence determination．The results showed：the full length of S4 is 3262bp，it contains one large open reading fram e an d encodes a polype ptide with 1058 amino acids in length．Tlle similarity of predicted protein were 94％ 、92％ an d 22％ with LdCPV、BmCPV an d RRSV respectively． Amino acid sequences simi lar to a GTPase、methyltran sferase of Methanosarcina mazef Goel motifs were also present in the deduced protein．
Abstract：Pseudelatia separata(Lepidoptera：Noctuidae)infected with Pseudelatia separata NPV (asNPV)and Pseudelatia separata GV (PsGV)and coinfected with the two viruses，the structural characters of its pi0thoracic gland( rG)and the ultrastructure of the gland cells were observed．The results showed that pathological chan ges appeared in the infected PTGs．In P lV infected treatment, the color of the glan d was purple—black dyed with eosin an d the cells were distorted ly squeezed together． The tracheae nearby showed grievous pathological chan ges an d a lot of white granules deposited around it．The pathological chan ges in PsGV infected treatm ent showed an other pattern．The glan d body was relative small with smaller individual cells，most of which were red dyed with eosin．The cell limits were obscure．In coinfected treatment．there were few glan d cells colorated by esoin．The ultrastructure observ ation also showed some pathological chan ges in the glan d cells
Abstract：A recombinant virus containing double copies of V—cath gene was constructed by using Han pvid Bac—to—Bac system．ThiS recombinan t virus was named as dciHaNP、 because it was inserted an other copy of V-cath gene controlled by iel promoter．Dot blot an d Northern blot an alysis showed that this recombinan t virus was correct．The toxicity was measured by using Hz—AM l cells infec ted with the recombinan t virus．Th e TCID5n ofdciHaNPlV is 3．16× l0 TCID5o／mL．
The full-length genome of Foot-and-Mouth disease virus (FMDV) OH99strain was cloned by RT-PCR and 3’RACE, and sequenced. The result shows that the full-length genome of OH99 strain is 8040nt in length, including 5’NCR, Leader protein coding region, Polyprotein coding region, and 3’NCR Their nucleotide sequence is 1026nt, 603nt, 6318nt, and 93nt respectively. Besides, poly (A) tail was also coined by 3’RACE, it’s 56nt in length. The full-length genome of OH99 strain was compared with other reference strains, and its genome characteristic is also analyzed. With the help of some biological ware, The Phylogenetic tree obtained from the nucleotide sequence of VPlgene from OH99 strain and ten reference strains shows that OH99 strain could be ranked FMDV O style. Furthermore, this strain and OTY TW/97 are in the same genotype, their nucleotide sequence share high homology, but it shows remarkable divergence with other reference strains, including china99 strain. For example, two short genome segment was deleted in t
According to the published IBV gene sequence, a pair of specific primers were designed and synthesized. A fragment of 1.7kb including mRNAs and mRNA_6 cDNA of infectious bronchitis virus LX4 was amplified, cloned and sequenced. Then the nucleotide and the deduced amino acid sequence of this fragment were analyzed. The results showed that N gene of IBV LX4 sharedthe highest identity with SD/97/02 and HB that were both isolated in China. However, the identity is different in three conservative basic regions of N protein. The deduced amino acid sequence of LX4 shared the highest identity with M41,Beau, H52 and HB in position 66-88, with SD/97/02 strain in position 181-234 and with H120 and ZJ971 in position 334-373 respectively. Meanwhile, we found that LX4 and H120 showed completely identical in position 350-409 both in corresponding nucleotide and deduced amino acid. All these data above showed that mRNA_5 and mRNA_6 cDNA of IBV LX4 shared more characteristics with other isolates in China than the aboard. In a
We jointly applied immunofluorescence technique, RT-PCR assay and flow cytometer to evaluate the stability of a cell model persistently infected by Classical swine fever virus (CSFV39), which was established by our laboratory and named CSFV39-PK15. The experiment results indicated that this cell model was quite stable. Even in the 128th CSFV39-PK15 passage cells, we still could detect the existence of CSFV39. Both immunofluorescence antibody test and RT-PCR assay showed positive results. We also found there was no significant difference in their cell cycle between normal control PK-15 and CSFV39-PK15. Sequence homology analysis of the same fragment revealed that CSFV39 had the highest homology compared with Shimen strain, amounting to 99.02%.
gB gene of Marek’s disease virus (MDV) was inserted into pcDNA3, forming eukaryotic expression recombinant plasmid pcDNA3-gB. Recombinant plasmid pcDNA3-gB was identified by BamHⅠI and PCR. PcDNA3-gB was transfected into COS-7 cells in vitro. Expressed protein was detected by indirect immunofluorescence assay. Immune test groups were inoculated with pcDNA3-gB intramuscularly. Two weeks later, test groups were boosted with the same pcDNA3-gB. Since then, two weeks later chickens were challenged with vMDV. Immune effects showed that the immune protection rate of test group reached to 72.2%. The results proved that pcDNA3-gB DNA immunization elicited immune protection.
The gene of nucleocapsid protein of Porcine reproductive and respiratory syndrome virus (PRRSV) VR2332 strain was amplified by RT-PCR. The PCR product was purified and digested with BamH Ⅰ and Xho Ⅰ, then directly cloned into the prokaryotic vector pET32a. Consequently the recombinant plasmid was constructed, designated pETN. PETN was transformed into the host cell BL21 (DE3) and the expression procedure was optimized including cultivated temperature, optional induction concentration and time of IPTG. The result indicated that the nucleocapsid protein can be expressed efficiently with 0.8mmol/L IPTG and 4-hour induction. The gene of nucleocapsid protein of Porcine reproductive and respiratory syndrome virus (PRRSV) VR2332 strain was amplified by RT-PCR. The PCR product was purified and digested with BamH Ⅰ and Xho Ⅰ, then directly cloned into the prokaryotic vector pET32a. Consequently the recombinant plasmid was constructed, designated pETN. PETN was transformed into the host cell BL21 (DE3) and the expression procedure was optimized including cultivated temperature, optional induction concentration and time of IPTG. The result indicated that the nucleocapsid protein can be expressed efficiently with 0.8mmol/L IPTG and 4-hour induction.
Both microcalorimetric measurement analysis by using LKB2277 bioactivity monitor and traditional method used in one-step growth curve in virology were carded out on BHK-21 cell line infected by FMDV (Foot-and-Mouth Disease Virus). The results show that microcalorimetric study can be applied in virus infection research through measuring metabolic energy released from BHK-21 cell and BHK-21 cell infected by FMDV. Meantime this microcalorimetric method provided a kind of new dynamic continuous analysis method on the virus infection research.
Abstract：A lot of spherical virions were observed in internal organiT．ation such as gill and mantellum of the diseased Mytilus edulis with negative staining method．The diameter of V on is around 8()_一 200nm with dual unit membran e．Sam e virions were observed in connective tissuese of infected organ s with ultrathin section electronmicroscopic observ ation．Th e“occlusion body’’were observed in cell matrix an d no inclusion bod y was present．A few same virions were observed in internal organization of normal Ruditapes variega~，Neptunea cumingi an d Dstrea lalienwhanensis with negative staining method．，nle normal Chlorostoma rusticum Can be infected by organizational liquid with the viruses from diseased Mytilus edulis through mouth and injured section on skin．Th e virus was purified from the diseased tissue by sucrose gradient centrifugation with 25 0oo r／min．3h．Th e virus sediment lies between 2O％ ～ 3O％ of sucrose． Key word：Mytilus edulis；SphericalAbstract：A lot of spherical virions were observed in internal organiT．ation such as gill and mantellum of the diseased Mytilus edulis with negative staining method．The diameter of V on is around 8()_一 200nm with dual unit membran e．Sam e virions were observed in connective tissuese of infected organ s with ultrathin section electronmicroscopic observ ation．Th e“occlusion body’’were observed in cell matrix an d no inclusion bod y was present．A few same virions were observed in internal organization of normal Ruditapes variega~，Neptunea cumingi an d Dstrea lalienwhanensis with negative staining method．，nle normal Chlorostoma rusticum Can be infected by organizational liquid with the viruses from diseased Mytilus edulis through mouth and injured section on skin．Th e virus was purified from the diseased tissue by sucrose gradient centrifugation with 25 0oo r／min．3h．Th e virus sediment lies between 2O％ ～ 3O％ of sucrose．
Abstract：The mosaic disease samples of Cucurbira pepo L．vail ov~ra and Cucumis melo L．vat． conomon were collected in greenhouse an d field at various sites in Congming Islan d．After host bioassay, electron microscopy(EM)observation，DAS—ELISA testing and molecular detection，the results showed that the pathogen of mosaic virus in e pepo L．var．ov~ra and C melo L．var．conomon is Zucchini yellow mosaic virus(ZYMV)．
Abstract：Cell membran e extracted from shrimp gill was coated on 96 well plate and bound witIl DIG labeled White spot syndrome virus(wssv)，then detected with Anti—DIG—Fab—HRP system，meanwhile taking the cell membran e from hepatopancreas，muscle，and E．coli for control group，the result verified the specific binding between W SSV an d gill cell membran e ．And here the binding assay can be employed as a convenient an d rapid way to evaluate the binding nature between W SSV an d the shrimp gill cell membran e．Abstract：Cell membran e extracted from shrimp gill was coated on 96 well plate and bound witIl DIG labeled White spot syndrome virus(wssv)，then detected with Anti—DIG—Fab—HRP system，meanwhile taking the cell membran e from hepatopancreas，muscle，and E．coli for control group，the result verified the specific binding between W SSV an d gill cell membran e ．And here the binding assay can be employed as a convenient an d rapid way to evaluate the binding nature between W SSV an d the shrimp gill cell membran e．
