2002 Vol.17(4)
The aim of this study was to prove the relationships of serotypes and nucleotide sequences of the Dengue viruses genome．To this end，the nucleotide sequences of 5 noncoding region，3 一noncod— ing region，envelope E glycoprotein，nonstructural protein NS1 were aligned correspondingly．W ith four Dengue viruses phylogenetic groups which were correspo nding to four serotypes each other being identified，Our results demonstrate that there were no intergenomic recombination observed．The po s— sibility of intragenomic recombination and the similarity of nucleotide sequences of different Dengue viruses were analysed．
Using molecular cloning technique，HPV16 E7 gene was cloned into eukarytic expression vector．HPV16 E7 DNA vaccine was constructed and Balb／c mice were immunized by intradermal ad— ministration with HPV16 E7 vaccine．After immunization，mice spleen lymphocytes were prepared and restimulated by E7 protein in vitro．Specific lympproliferation were detected by MTT colorimetric as— say．Because detection of specific lymphoproliferation is a simple and efficient way，which can reflect cell—mediated immunity．The study showed that the construction of HPV16 E7 vaccine was correct．It can induce a specific cell—mediated immunization
In order to use major capsid protein L1 of Human papillomaviruses(HPV)16 produced in a fused fom in E ．coli and HPV16 L1 VLP produced in recombinant adenovirus in 293 cells as antigen to detecton antibodies of HPV 1 6 L1 in the cervical cancer people．and compare the serological differ— enee of the two antigen in the diagnosis of cervical cancer，we used PCR to amplify HPV1 6L1 gene from the cervical cancer， then cloned into pUC18一T． After DNA sequencing， HPV16L1 gene was cloned into pGEX一2T expressing vector，and induced by IPTG to express in E ．coli as glutathione—S— transferase—L1(GST—LI)fusions and purified to near homogeneity as antigen，together with HPV16 L1 VLP produced in recombinant adenovirus in 293 cells，to test antibo dies of human—papillomavirus (HPV)16 L1 of 12 cervical cancer and 53 blood donors．The gene derived from the cervical cancer HPV16 genome was 1535 bp in length，and expressed by E ．coli to the full—length 83 kD polypep— tide，which recognized 1)y HPV16L1 monocloned antibody．In the 12 cervical cancer sera，there were 7 positive in HPV16L1 antibody(58．3％ )；while 8 positive in HPV16L1 VLP antibody(66．7％)．A— mong 7 positive in anti—HPV16 L1using HPV16L1 protein from the E ．coli as antigen，all are positive when using HPV 16L1一V LP as antigen．W hile among 5 negative in anti—HPV16 Llusing HPV 16L1 protein from the E ．coli as antigen， there is 1 positive when using HPV1 6L1一VI P as antigen．There are no difference between two group as antigens in ELISA detection．(P0．05)Our research sug． gested that HPV 1 6 is highly associated with cervical cancer．The sensitivity of the test is the same whether using HPV 16I 1 protein from the E ． coli or HPV16L1一VLP as antigens． To detect HPV16I 1 antibody is helpful to diagnosis cervical cancer．
To construct a recombinant eukaryotic expression vector bearing fusion gene Of Hepatitis B viruS(HBV)S2+S gene and IFN-a gene，technique of splicing by overlapping extension and two times PCR were used．Fusion gene fragment was obtained and directly cloned into pcDNA3．1 V5／His TOPO TA cloning vactor to get recombinant eukaryotic expression vector pcDNA3．1$2S／IFN—a． Then the recombinant vector was transferred into Vero E6 cells using LipofectAM INE．The recombi— nant vector was constructed and correctly checked by digestion with restriction enzymes and poly— merase chain reaction．The vector bearing fusion gene could be expresed in eukaryotic cells detected by indirect immunofluotescence technique．The relative efficient expression of the fusion gene in Vero E6 cells might provide an experimental basis for specific immunotherapy for HBV infection．
The influenza epidemic situation in the epidemic year of M arch 200 1 to March 2002 in Hubei area was analyzed according to the results of isolations of Infzuenza viruses，detection of human antibodies against Infzuenza viruses and epidemiological survey．It was concluded that there were two epidemics during the epidemic year．One in August and September of 200 1 was mainly caused by A1 type of Infzuenza viruses．Another was caused by the dominant strains of A3 type．It was also shown that the isolated strains of A3 type during this epidemic year likely underwent some antigenic draft， compared with the A3 type of Infzuenza viruses isolated in 1 999．
A 8．5kb fragment containing an E ．coli low—copy number mini F replicon，a selectable kanamycin resistance marker and lacZ gene with attTn7(the target site for bacterial transposon Tn7) replaced polyhedrin gene in HaSNPV genome using homologous recombination．HaSNPV genome was cloned and maintained as 1 32 kb bacterial artificial chromosome(Bacmid)in E．coli，transfection of the Bacmid(HaBacmid．HZ8)into HzAml cells led to a productive virus infection．In this paper．the donor plasmid HapFastPhP10 was constructed using the Ha—polh gene and the Ha．P10 promoter tO re． place the original Ac P1 0 promoter and Ac—Polh promoter of the pFastBacDual donor plasmid respec． tively．The eGFP gene was inserted into multiple cloning site(MCS)downsream of the P10 promoter in the pFastBacHaPhpP10 donor plasmid．Recombinant HaBacmid HZ8 was constructed by transpo s． ing a mini—Tn7 element from a HaDFastPhP1 0 donor plasmid tO the mini．attTn7 attachment site on the HaBacmid—HZ8 when the Tn7 transposition functions are provided in trans by a helper plasmid．Re． combinant HaBacmid．HZ8 DNA was transfected HzAml cells．occlusion bodies and green flusecent were found within HzAml cells 5 days after transfection．The results indicated the Habac to Bac ex． pression system based on HaSNPV can effectively express foreign gene．
Enhancin is a group of baculovirus proteins capable of increasing the infectivity of viruses in insect larvae，as well as the insecticidal effect of other biology control agents． In order to study the structure and function of enhancin from Trichoplusia i gran ulovirus， recombinant baculoviruses were constrcuted to express truncated forms of enhancin with 150， 186 and 250 am ino acids deleted from N terminal of native enhancin，respectively．The truncated enhancins were expressed successfully in Tn一5B1—4 cells． In vitro peritrophic membrane assay showed that all three truncated enhancin lost their mucin degrading ability，while a full length recombinant enhancin was active in the assay．It is concluded that the N terminal amino acids is essential for the mucin degrading activity of enhancin．
The PCR product of the HaSNPV chitinase gene，which was without the N—terminal signal peptide and the C-terminal endoplasmic reticulum location signal，was cloned into a prokaryotic expres— sion vector pProEXHTb．After the induction of IPTG，the chitinase gene was successfully expressed in Eschechia coli DH5a．The expression product shown a molecular weight of 60 kDa，and it covered about 40％ of the total protein in E ．coli．W estern blot analysis using an antibody derived from AcM — NPV ehitinase confirm ed the expression product was a homologue of baculoviral chitinase．
A recombination transposing vector pBlueBacHis2一CagA was constructed by inserting Heli— cobacter pylori f口gA gene into pBlueBacHis2A vector of baculovirus expession system ．Co—transfecting Sf9 cells with Bac—N—blue DNA and pBlueBacHis2一CagA，recombinant viral DNA was obtained by pu— rification of recombinant plaques．PCR result showed ca gene intergrating in correct position of bac— ulovirus gemone．The expression of Helicobacter pylori ca gene was confirmed by SDS-PAGE and W esternbolt．Using indirect ELISA assay，it was found that the protein exhibited good specific reac— tivity with sera of HP—infected individuals
Two cDNA fragments encoding human interleukin一12(hII一12)P35 and P40 subunits were isolated by RT．PCR from KB cells stimulated with PDBu，and then cloned into pCR ．1 respcetively and down the double promoters of pAcUw 5 1．The recombinant baculovirus Ac—hiL2 was obtained by cotransfecting with pAeUW 5 1一ILl2 and baeuloGold M Linearized baculovirus DNA．Argyrogramma agnata larvae were infected with recombinant baculovirus Ac—hiLl2 by hemoeoel injection．The rhII， 1 2 was purified from the supernatant with affinity chromatography．The blood lymph supernatant har— vested and rhII，12 purified were SUbjected to SDS-PAGE(silver stain)and Western blot．The level of rhIL-12 was detected by EU SA．Bioactivity of purified rhII，12 samples were detected by M 1vr method．It is indicated that Ae-hlL12 can replicate in fat body and midgut cells of Argyrogramma Agnata larvae．The MW of rhlL一12 expressed was 75kD．The expresion levels of rhIL-12 were 17．8 ／~g／lO cells and 200—300mg／L in Sf9 cells and Argyrogramma agnata Staudinger larvae blod lymph respectively．The purified rhII，12 has significant activities which enhanced NK eytotoxieity and increased the proliferation of human PBMC PHA-P activated significantly．
Homology and phylogenetic tree analysis of genus Bymovirus suggested that the 5 一UTR of RNA1 and RNA2 of the same virus were more similar than those of the same RNAs of different virus— es．but the instance of 3 UTR was opposite．Secondary structure analysis on polypoteins showed that P3 and 14K encoded by RNA1 contained transmembrane(TM)stuctures．TM region in P3 protein of BaM MV ALS1 isolates was toxic to the E ．coli．By analogy to other members of family Potyviridae． these structures were predicted to have the function as attachment to the membranes．P2 protein en— coded by RNA2 also contained two TM regions．Some BaMMV isolates which were maintained me— chanically for a long period of times occurred deletions in these regions and were not transmitted by Polymyxa graminis．It was suggested that the TM structures were closely related to vector transmis— sion．
A antiviral protein。YP46—46，was isolated and purified to homogeneity from the mushroom， Plearotus citrinopileatus．by precipitation of 40％ 一60％ saturation of ammonium sulfate followed by DEAE-sepharose FF ion—exchange column chromatography。 Sephacryl M S一200 High Resolution molecular sieve chromatography．The protein has a molecular weight of about 27．4kD by SDS-PAGE． The concentration of the protein was 0．24／~g／mL when the inhibition rate against Tobacco mosaic irus was 50％ ．Using an assay system based on HepG2．2．2．1 5 cell line，YP46—46 was studied for its inhibitory ability against Hepatitis B virus．The result shows that the concentration of 50％ inhibition of HBsAg was 0．08ttg／mL，but low effect on HBeAg．
Bovine immunodeficiency virus(BIV)is a kind of Lentivirus belonging tO Retrovirus，and up tO now．there is no report about itS infection tO human．In order to check BIV S infection to human cells，we transfected BIVl27 cDAN into human cells MT一4．By RT～PCR，we checked the transcription of BIV S gag gene，and determined the expression of gag or gag-pol gene in M T一4 cell by IFA．Re— verse Transcriptase(RT)assay showed the reverse transcriptase of BIVl27 had been expressed，while cell infection experiment indicated that BIV couldn t replicate in MT一4 cells．
The methods of reverse transcription，polymerase chain reaction(PCR)amplification，and cloning of full—length VP2—4—3 gene of a very virulent infectious Bursal disease virus(vvlBDV)strain SH95 were developed．The use of random primer and a reverse transcriptase lacking RNase—H activity produced full—length coding region and non—coding region eDNA copies of the viral genomic segments． The 3060 base—pairs(bp)of VP2—4—3 were amplified by long and accurate PCR in a single step，SUC— cessfully cloned and sequenced revealing their identity of IBDV．
Bluetongue virus(BTV)HbC strain and BTV 10(the international standard strain)were respectively inoculated onto the different species monolayer cells such as Vero cell(monkey renal cel1)， C6 cell(mouse neuroglioma cel1)and Hela cell(human cervical cancer cel1)．We studied comparatively the replicated characteristics of BTV—HbC in different species cells，with BTV 10 in the same cells， morphologic characteristics under microscopy and electron microscopy after interaction between BTV— HbC and different cells．the serologic relation between BTV—HbC and BTV一10．It is further proved that the BTV—HbC strain is probably a new type of Bluetongue virus when connecting our previous re— search about BTV—HbC genome map and RT—PCR analyses of BTV—HbC gene coding group speci fic antigen．
Two new methods，colony counting method and plate inducing method，were established for determining ultraviolet induction rate of prophage in the present study．Culture of lysogenic bacteria (λ)which was induced hy ultraviolet irradiation and cultivated in dark was directly spread on the plate，and induction rate of prophage was calculated based on the CFUs(colony form ing units)．The plate with mixture of lysogenic bacteria and indicator strain was induced by ultraviolet irradiation．The induction rate of prophage was calculated based on the PFUs(plaque form ing units)．These two methods not only can determ ine precisely induction frequency of prophage。but also are easy tO ma— nipulate ，and they can save efforts and apparatus，they are also easy to repeat．Furthermore，the m ix— ture of Cordyceps sinensis extract and lysogenic bacteria was irradiated with U V ．The results demon— strate that the new methods are practical and easy tO conduct，and that Cordyceps sinensis has a strong protection effect against U V—irradiation．
We investigated the infection of Avian leukosis virus subgroup J myelocytomatosis usingEI ISA method in Inner Mongolia Region．The result indicated that the meat—type flocks of three dif—ferent farms infection rate'．of ALV—J were 28．12％(9／32)、9．38％(3／32)and 8．33％(10／12)，respec—tively，and all laying chickens infection rate were 0％(0／35，0／10)．We chose 6 chickens of ALV—J an—tibody positive and 13 chickens of ALV—J antibody negative to do PCR test．The result indicated that6 chickens of ALV—J antibody positive were all PCR positive，and 4 chickens were PCR positive among1 3 chickens of AI V—J antibody negative．The result indicated that there were ALV—J virus not only inAI V—J antibody positive chickens but also in some AI V—J antibody negative chickens．
Several methods were t~ted for measurement of lysing cycle and burst size of the cyanophage infecting the ilia— mentous cyanobacterium—Plectonema boryanum IU594．The rm~ts indicated that the lysing curve of host cells was con— sistent with the one-step growth curve of cyanophage，thus it was more convenient tO substitute measuring the lysing curve of host cells for the one—step growth curve measurement of cyanophage．Besides，the burst size increased with the re— ducing cyanophage inoculation．reaching the maxium of 206PFU／Cel by 1 PFU infection．It was suggested that more ex— act burst size of cyanophage could be obtained under 1PFU cyanophage inoculation．
分子伴侣(molecularchaperone)的概念，由Lasky于1978年首先提出，真核细胞内质网中的分子伴侣是由多种蛋白质组成，它 们可以介导新合成蛋白质的正确折叠与装配，并在真核生物的细胞中广泛存在。
猪流行性腹泻(porcine epidemic diarrhea，PED) 是以水泻、呕吐和脱水为特征的一种急性病毒性腹 泻。猪流行性腹泻现已成为世界范围内的猪病之 一 。猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)是PED的致病因子，是导致类似猪传 染性胃肠炎(porcine transmissible gastroenteritis， TGE)临床症状的真正病原。迄今为止已发现 PEDV与TGEV[ 、PEDV与PCV混合感染猪【 。 已有用蛋黄IgY预防PED效果的报道L3 J，还有用弱 化的PEDV疫苗对仔猪进行免疫的报道 ]，但都对 其作用机制未作深入探讨。弄清PEDV 的分子生 物学特征，针对PED进行特异性免疫，必将对PED 的诊断、治疗和综合防治产生深远影响。本文仅就 PEDV的分子生物学特征作一综述。
Coltivirus病毒属(Coltivirus，Colti)为呼肠病 毒科病毒，以科罗拉多蜱媒热病毒(Colorado tick fever virus，CTFV)为代表株，该病毒原为环状病毒 属成员，由于其对人有致病性，可引起人的发热和脑 炎，病毒核心的衣壳表面结构不同于典型的环状病 毒，尤其是病毒基因组为12个双链RNA节段而不 是环状病毒的10个节段，核酸分子量27×10 ，远 比环状病毒(18×10 )大，故单列一属。目前全世界 已在美洲、亚洲和欧洲等地分离到数十株Colti病毒 (主要病毒株的分离年代和地区见表1)。其中CT— FV 和在德国、法国分离到的Eyach及其变异株 Ar577和Ar578被称为欧美流行株，亚洲分离株除 印尼的JKT6423、JKT6969、JKT7043、JKT7075分 离株外，我国自1980年以来也从人、畜标本和野外 采集的蚊虫及蜱标本分离到多株Colti病毒，如目前 研究较多的有云南分离株Banna病毒，北京分离株 BJ95—75、BJ95—70、TRT2，东北分离株NE97— 12、NE97—31等，这些病毒与科罗拉多蜱媒热病毒 在生物学特性及致病性等均存在较大差异。本文就 Colti病毒的生物学特征、对人、畜的致病性与流行 病学等进行了总结，特别是近年来国内外注重对 Colti病毒的分子生物学特征研究，获得较多进展， 由于病毒基因组已被阐明，对许多Colti病毒的生物 学特性可以用分子生物学加以解释，作者将结合本 人和国内的工作做一综述。
